Package for plant antimicrobial treatment

ABSTRACT

The present invention provides a package ( 200 ) comprising at least one first component in the form a well ( 220 ), the first component comprising particulate matter comprising at least one natural oil; at least one second component, in the form of a plurality of second wells ( 230 ) comprising antagonist(s) of a microbial pathogen; wherein the at least one first component and the at least one second component are contained in separate compartments of said package. Also provided by the present invention is a method for providing an anti- bacterial agent, the method comprising mixing the first component comprising particulate matter carrying at least one natural oil; and at least one second component comprising at least one antagonist of a microbial pathogen, and allowing said mixture to form into an emulsion with anti-bacterial activity. Further provided by the present invention is a method of treating or preventing a pathogen infection in a plant, the method comprises applying to said plant an amount of an emulsion comprising particulate matter, at least one natural oil and at least one antagonist of a microbial pathogen that causes said pathogen infection. Yet further, there are provided some isolated antagonistic bacteria that may be used, inter alia, in the package and methods disclosed herein.

TECHNOLOGICAL FIELD

The present disclosure is in the field of products for antimicrobial useand in particular, plant protection and biological control of plants.

PRIOR ART

References considered to be relevant as background to the presentlydisclosed subject matter are listed below:

US patent application publications No. US2011028500

US patent application publication No. US2011014596

Indian Patent Application No. IN03603CH2010

U.S. Pat. No. 7,485,451

Ait Ben Aoumar A. et al. J. Med. Plants Res. 6(17):4332-4338, 2011

Pouvova D. et al. Zemdirbyste-Agrucyktyre 95(3):440-446, 2008

Lanteigne C, et al. Phytopathology.102(10):967-73, 2012

Slusarski C. Vegetable Crop Research Bulletin 69:125-134 2008

Acknowledgement of the above references herein is not to be inferred asmeaning that these are in any way relevant to the patentability of thepresently disclosed subject matter.

BACKGROUND

Agricultural crops are susceptible to a large variety of microbialpathogens, which results in annual losses and economical damages.Methods developed to protect crops from plant diseases include plantbreeding for resistance, cultural practices, application of chemicalagents, and biological control.

As the use of chemical pesticides resulted in severe environmentalpollution, and many pathogens are developing resistance to existingchemicals, many pesticides are now banned for use, and organic farmingis not allowed to rely on such substances at all. Thus, a major goal,therefore, is to develop new, environmentally-friendly tools to controlpathogens, namely, biological control techniques.

U.S. Pat. No. 6,495,133 describes a strain of Glicladium roseumexhibiting antagonistic effects against plant pathogens. The biocontrolagent is used in treatment o seeds, soil or plants to protect againstfungal pathogens of various plants, including tomato.

US patent applications publications Nos. US2011028500 and US2011014596describe a plant pathogen inhibitor combination comprising a plantextract containing one or more anthraquinone; an anti-phytopathogenicagent which may include natural oil or oil product having fungicidalactivity.

Indian Patent Application No. IN03603CH2010 describes an invert-emulsionformulation of fungal organisms as biological control. The formulationis in the form of an invert emulsion formulation. The process describedincludes production of fungal spores either by solid state or liquidfermentation; preparation of conidial suspension or cell suspension;preparation of aqueous phase by mixing the conidial suspension, water,emulsifier and glycerol; preparation of oil phase by adding vegetablefat mixture to a warm vegetable oil mixture; mixing of aqueous phasewith oil phase to get water in oil or invert-emulsion formulation usinghomogenizers The product can be used for seed treatment, soilapplication and foliar spray.

U.S. Pat. No. 7,485,451 also describes an invert emulsions (water in oilemulsions) comprising cellular material selected from living and/ordormant prokaryotic and/or eukaryotic cells and tissues, the cellularmaterial being compatible with water-in-oil emulsions. Examples ofcellular material included fungi, watermolds, algae, yeasts, bacteria,plant, inset and animal cells. The inverted emulsion also comprises anoil, such as vegetable oil and/or fish oil as well as an oil solublenon-ionic surfactant, and water. Optionally, the composition contains athickener, such as fumed silica or bentonite.

Essential oils having an antagonistic effect have also beeninvestigated. For example, the antibacterial activity of Moroccan plantsextracts against Clavibacter michiganensis subsp. Michiganensis (CBM),the cause of tomato bacterial canker, was described [AA Ben Aoumar A. etal. J. Med. Plants Res. 6(17):4332-4338, 2011]. In addition, theeffectivity against CBM of plant essential oils from 34 aromatic plantswas examined [Pouvova D. et al. Zemdirbyste-Agrucyktyre 95(3):440-446,2008].

Recently, the simultaneous production of DAPG and HCN by Pseudomonas sp.LBUM300 was found to be beneficial for the biological control of tomatobacterial canker caused by Clavibacter michiganensis subsp.michiganensis [Lanteigne C, et al. Phytopathology. 102(10):967-73,2012].

In addition, attempts at biological control of CBM Rockwool-growngreenhouse tomatoes was described. Specifically, artificial inoculationof two and three years old rockwool slabs with CBM bacteria dead plantsreduces death rate of the plants [Slusarski C. Vegetable Crop ResearchBulletin 69:125-134 2008].

General Description

Biological control of plant diseases generally is defined as suppressionof pathogens by application of one or more organisms that exhibitantagonistic activity towards the pathogens. The organisms that act asantagonists are regarded as biological control agents (BCAs) and themechanisms of the antagonistic effects are based on a variety ofbiological properties of BCAs. These comprise production of antibioticcompounds, expression of enzymes that catalyze the decomposition of cellcomponents of pathogens, competition for space and nutrients, theability to parasitize pathogens, and the induction of plant defense.

The present disclosure is aimed at providing a ready for use package forbio-control of crops against microbial infection as well as forpreventing such infection from developing. As will be evident from thefollowing description, there are provided packages such that thebio-control components are isolated from each other during long termstorage and are easily mixed, at the pre-determined concentrations, uponneed, without any risk of environmental contamination by the package'scomponents. It has been found that the package configuration ensureschemical stability of the components, i.e. without any damage after longterm storage.

Accordingly, and in accordance with a first of its aspect, the presentdisclosure provides a package comprising:

-   -   at least one first component comprising particulate matter        comprising at least one natural oil;    -   at least one second component comprising at least one antagonist        of a microbial pathogen;        wherein the at least one first component and the at least one        second component are contained in separate compartments of said        package.

In some embodiments, the compartments are defined as wells within acarrier element forming part of the package.

According to some embodiments, the package comprises:

-   -   (i) one or more first wells (compartment) holding a first        component comprising particulate matter comprising at least one        natural oil;    -   (ii) one or more second wells (compartment) holding a second        component comprising at least one antagonist of a microbial        pathogen;

the one or more first wells and the one or more second wells each havinga top opening and a recess extending downwardly from said top opening,the wells being held together in an essentially planar matrix; and

-   -   (iii) a first film sealing the openings of the one or more first        wells; and    -   (iv) a second film sealing the openings of the one or more        second wells.

In accordance with a second of its aspects, there is provided a methodfor providing an anti-bacterial agent, the method comprises mixing oneor more first components comprising particulate matter, the particulatematter carrying at least one natural oil with one or more secondcomponents comprising at least one antagonist of a microbial pathogen,and allowing the mixture (cocktail) thus obtained to form into anemulsion. The thus formed emulsion poses anti-bacterial activity. Theemulsion prepared by the components disclosed herein is stable emulsion,i.e. where no phase separation is apparent for at least several hours,for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and even up to 24 hours.

In some embodiments, the method makes use of the packages disclosedherein. In some embodiments, the use of the package provides acomposition or an emulsion comprising particulate matter, a surfactant,at least one natural oil, and at least one bacterial antagonist of aplant pathogen. In some embodiments, the antagonist is of a time that iscapable of growing on sesame oil as a sole carbon source.

In accordance with yet a third of its aspects, the present disclosureprovides a method of treating or preventing a pathogen infection in aplant, the method comprises applying onto said plant an amount of anemulsion comprising particulate matter, at least one natural oil and atleast one antagonist of the pathogen that causes the infection.

In accordance with a fourth of its aspects, the present disclosureprovides isolated antagonistic bacteria having a representative sampledeposited at the CBS-KNAW institute and bearing the accession No.selected from the group consisting of CBS133252, CBS133254, CBS133255,CBS133256, CBS133257, CBS133258, CBS133259, CBS134566, CBS134567 andCBS134568. In some embodiments, these antagonistic bacteria are for usein protecting or treating plants against a pathogen induced infection.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIGS. 1A-1D show a carrier element forming part of a package accordingto an embodiment of the invention, from different views, with FIG. 1Aproviding an isometric view, FIG. 1B providing a top view and FIGS. 1Cand 1D providing views from side X and side Y of FIG. 1B.

FIGS. 2A-2B show views of a carrier element in accordance with FIGS.1A-1D, including films sealing the different wells of the carrierelement, in accordance with an embodiment of the invention.

FIGS. 3A-3E show a carrier element according to an embodiment of theinvention, from different views with FIG. 3A providing an isometricview, FIG. 3B providing a top view and FIGS. 3C, 3D and 3E, providingviews from sides Z, X and Y of FIG. 3B.

FIG. 4A-4C show the effect of two selected antagonists on growth of CBM(FIG. 4A) and Xanthromonas (FIG. 4B) as compared to control (FIG. 4C).

FIGS. 5A to 5E show petri dishes of various pathogens after treatmentwith a plant pathogen, with or without an antagonistic bacteria

FIG. 6 is a graph showing the mortality rate of tomato plants followingexposure to Clavibacter michiganensis subsp. Michiganensis (CBM), theplants were either treated with combinations according to someembodiments of the present disclosure, or with controls beforeinoculation with CBM.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention is aimed at providing a package that is suitablefor long term storage and upon need, provides a safe and easy method forpreparing anti-microbial compositions for various applications, such asfor crop protection. To this end, a package has been developed, havingtwo (or more) wells or set of wells, each well or set of wells carryinga different component. The package is configured such that the two (ormore) wells are separately and differently sealed, due, inter alia, todifferent physical and chemical characteristics of the matter in thecompartments, as will be further discussed hereinafter.

Generally, the package comprises:

-   -   at least one first component comprising particulate matter        comprising at least one natural oil;    -   at least one second component comprising an antagonist of a        microbial pathogen;    -   wherein the at least one first component and the at least one        second component are contained in separate compartments of said        package. In other words, as long as the kit is sealed, the two        components are not mixed or are not in contact with each other.

Upon mixing of the first and second components (i.e. taking out the twocomponents from their separate compartments), a stable emulsion isobtained, suitable for application onto the crop to be treated orprotected. The emulsion is applied in a form of fine droplets.

In the context of the present disclosure, the term “particulate matter”is used to denote a substance in the form of plurality of particle. Theparticles may be in any particulate form, including, without limitedthereto, from finely rounded beads to amorphous structures. Theparticulate matter includes any form of a powder.

In some embodiments, the particulate matter comprise silica dioxide(SiO₂, in short referred to herein as silica). The silica may benaturally occurring silica particles such as bentonite clay beads, aswell as synthetic silica beads.

In some embodiments, the particulate matter comprises synthetic silicaparticles. There are a variety of synthetic silica particles that may beused in the context of the present disclosure. For example, theparticulate matter may comprise precipitated synthetic amorphous silicabeads, such as the commercially available products Tixosil and Aerosil200.

In some other embodiments, the particulate matter comprises synthetic ornature derived beads with the capacity to absorb the natural oils. Suchbeads may include, without being limited thereto Latex beads; calciumcarbonate sorbent particle; cellulose beads; polystyrene adsorbentsbeads e.g. Amberlite® XAD®-2 which is a hydrophobic crosslinkedpolystyrene copolymer absorbent resin; charcoal; Sepharose™ beads;emulsan-alginate beads; chitosan beads; sodium alginate; styrene-maleicacid copolymer beads and styrene-divinylbenzene beads; cellulose paperbeads.

To allow good distribution of the final emulsion and in accordance withsome embodiments the particulate matter (particles) has a sizedistribution in the range of 10-25 μm.

The particulate matter may also be characterized, without being limitedthereto, by one or more of a surface area, in some embodiments, in therange of 400-500 m² N₂/g and oil capacity in the range of 300-350DBP/100 gram particulate.

The first component comprises the particulate matter that holds one or acombination of natural oils. In the context of the present disclosure itis to be understood that “natural oil” encompasses any organic oilobtained from nature.

The natural oil is preferably oil derived from a plant. In someembodiments, the natural oils are known as essential oils. The essentialoils are preferably those known to exhibit antimicrobial (e.g.antibacterial, antifungal, antinematodal) properties.

When referring to anti-microbial properties it is to be understood asbeing effective against any microbial pathogen, as further discussedbelow.

Without being limited thereto, essential oils to be used in accordancewith the present disclosure, may be those derived from the plantsOriganum vulgare and Origanum spp., (e.g. Oregano), Mentha spp. (mint),Thymus spp. (Thyme), Myrtus spp., Ocimun spp. (e.g. Ocimun basilicum,also klnown as Basil), Lavandula spp. (e.g. Lavender), Micromeria spp.,Coriandum spp. (e.g. Coriander/Parsley), Aloysia spp., Melissa spp.,Salvia spp., Petoselinum spp., Rosmarinus spp. (e.g. Rosemary), Prunellaspp., Cuminum spp (e.g. Cumin).

In some other embodiments, the natural oils are plant derived oils thatis used as carbon source, e.g. as food/nutrient for the antagonisticmicroorganisms. These are referred to herein the term “carbon-base oil”or “carbon-rich nutrient oil”. In some embodiments, the carbon-base oilsare vegetable oils. Without being limited thereto, the carbon-base oilis selected from the group consisting of Sesame oil, Olive oil, Peanutoil, Cottonseed oil, Soybean oil, Palm oil, sunflower oil, saffloweroil, canola oil, castor oil, coconut oil, groundnut oil.

In some embodiments, the term “natural oil”, when used in plurality,encompasses a combination of at least one essential oil and at least onecarbon-base oil, both being of natural source.

In some embodiments, the natural oil comprises at least Oregano oil incombination with at least one carbon-base oil. The Oregano oil iscombined, at times, with at least Sesame oil.

It has been unexpectedly found that the antagonistic bacteria may bedistinguished from other bacteria with no exhibited antagonisticactivity towards at least CBM by their capability to grow on carbon baseoil, such as sesame oil. In one embodiment, the carbon base oil on whichall antagonistic bacteria grow (while non-antagonistic bacteria testeddo not) is sesame oil.

The amount of the natural oil within the first component (e.g. held bythe particulate matter) may vary, depending on the type(s) of thenatural oil used, the amount at loading, the type of particulate matter,the conditions of loading the natural oil onto the particulate matter,the surfactants or solvents used for loading etc.

When referring to loading of the oil onto the particulate matter, it isto be understood as meaning any form of association between the oil andthe particulate matter (e.g. silica particles). Without being limitedthereto, the oil is held by the particulate matter by absorption ontoand/or into the particles. The association between the particles and theoil is reversible, namely, under suitable conditions, such as whenbrought into contact with water, the oil is easily released from theparticles to form an emulsion.

In some embodiments, the particulate matter holds between 20% to 50% w/wnatural oil out of the total weight of the particulate matter (afterloading). This is determined by conventional techniques such as HPLC orGC chromatography, as also exemplified below. In some other embodiments,the particulate matter holds about 30% w/w natural oil (“about”encompasses a range of between 25-35%, at times between 28% to 32% oraround 30%).

In some embodiments, the natural oil comprises either only the essentialoil(s) or a combination of at least one essential oil and at least onecarbon-base oil. As such, when referring to natural oils it is to beunderstood as encompassing essential oil(s) as well as carbon-baseoil(s). The ratio between the at least one essential oil and at leastone carbon-base oil is in the range of 60:40 and 100:0, at times therange is about 80:20.

When a combination of oils is used it is to be understood that they maybe absorbed onto the particulate matter together, i.e. the sameparticulate matter holds more than one type of oil. In some embodiments,for ease of handling, each oil type is held separately on particulatematter such that different types of particulate matter are formed, eachbeing characterized by the type of oil it is holding.

Thus, when referring to particular matter providing Oregano and Sesameat a ratio of 80:20 it is to be understood as a mixture of twopopulations of particulate matter, 80% carrying Oregano oil and 20% of atype carrying Sesame oil or a single population of particles, eachparticle being absorbed with the two oils at the defined or desiredratio (i.e. the oils are a priori mixed and then brought into contactwith the absorbing carrier/particle). Irrespective of the oil type, theparticulate matter between 20% to 50% w/w of its total weight itprovided by the oil loaded thereon.

The particulate matter may also comprise at least one surfactant. Asappreciated, a surfactant is a compound that lowers the surface tensionof a liquid and as such, the interfacial tension between two liquids toallow the formation of, e.g. an emulsion. The surfactant may be of anykind known in the art as safe for use (e.g. non-toxic to plants oranimals), including ionic surfactants, anionic surfactants, cationicsurfactants as well as zwitterionic (or non-ionic) surfactants.

In some embodiments, the surfactant is of a type acceptable in organicagriculture. A non-limiting list of possible surfactants to be used inaccordance with the present disclosure includes Polyethylene glycolsorbitan trioleates (Tween, e.g. Tween 85, Tween 65), sorbitan fattyacid esters (e.g. Span 40).

In some other embodiments, the surfactant comprises a salt of a fattyacid. The salt may comprise an alkaline such as potassium, calcium,sodium salts, as well as an ammonium salt.

In some embodiments, the salt of a fatty acid comprises potassium saltsof fatty acids (also known as soap salts), which are at times used asinsecticides, herbicides, fungicides, and/or algaecides. In someembodiments, potassium salts of fatty acids may be obtained by addingpotassium hydroxide to natural fatty acids such as those found in animalfats and in plant oils. Fatty acids may be extracted from olives, cottonseeds, soya beans, peanuts, sun flowers, coconuts Palm, Rapeseed, Sesameoil, Amaranth, Corn, Jatropha.

The fatty acid forming the surfactant may also be a synthetic fatty acidas well as a semi-synthetic (e.g. a natural fatty acid that underwent amodification).

In accordance with some embodiments, the at least one surfactant is onebeing recognized or is labeled as having an insecticide and/or fungicideactivity. Without being limited thereto, pesticidal and/or fungicidalsurfactants may include, the commercial products Zohar PT-50 and ZoharLQ-215, both produced by Zohar Dalia, Israel.

In one particular embodiment, the surfactant is selected from ZoharPT-50 and Zohar LQ-215.

The compositions of these surfactants are available from Zohar Dalia.For instance, Zohar PT-50 is known to have the following composition:

Vegetable oils Polyunsaturated fatty acids Mono- linolenic LinoleicOleic Saturated unsaturated Total acid acid acid Smoke Type fatty acidsfatty acids poly (ω-3) (ω-6) (ω-9) point Not hydrogenated Canola 7.36563.276 28.14  9-11 19-21 — 204° C. (rapeseed) Coconut 91.00 6.000 3.000— 2 6 177° C. Corn 12.948 27.576 54.67 1 58 28 232° C. Cottonseed 25.90017.800 51.90 1 54 19 216° C. Flaxseed/ 6-9 10-22 68-89 56-71 12-18 10-22107° C. Linseed (European) Olive 14.00 72.00 14.00 <1.5  9-20 — 193° C.Palm 49.300 37.000 9.300 — 10 40 235° C. Peanut 16.900 46.200 32.00 — 3248 225° C. Safflower 8.00 15.00 75.00 — — — 210° C. (>70% linoleic)Safflower 7.541 75.221 12.82 — — — 210° C. (high oleic) Soybean 15.65022.783 57.74 7 50 24 238° C. Sunflower 10.100 45.400 40.10 0.200 39.80045.300 227° C. (<60% linoleic) Sunflower 9.859 83.689 3.798 — — — 227°C. (>70% oleic) Fully hydrogenated Cottonseed 93.600 1.529 .587 .287(hydrog.) Palm 47.500 40.600 7.50 (hydrogenated) Soybean 21.100 73.700.400 .096 (hydrogen.) Values as percent (%) by weight of total fat.

The results provided herein show that a salt of a fatty acid asdisclosed herein had some advantage in terms of stability and/oremulsification properties of the powder, and anti-microbial activity,over other known surfactants, such as the commercially known Tween 20 orTween 80.

The amount of the surfactant in the first component may vary. However,in some embodiments, the particulate matter comprises between 5% to 10%w/w of the surfactant or combination of surfactants.

The first component comprising the particulate matter is in anessentially dry form. When referring to “essential dry” it is to beunderstood that the first component may contain low amounts of water, insome embodiments not more than 10% (w/w). In some other or additionalembodiments, the water content in the first component is within therange of 1% to 7% (w/w). In yet some other embodiments, the “essentialdry” is to be understood as encompassing no water being detected byconventional methods (i.e. no detectable amount of water).

The first component may also contain some trace amounts of an organicsolvent. As will be further discussed below, a solvent may be requiredfor the preparation of the particulate matter and some residual amountsmay remain, as long as the solvent is not toxic. In some embodiments,the first component is either solvent free (i.e. contains no detectableamounts of an organic solvent) or comprises trace amount, i.e. not morethan 5%, 4%, 3% or even 2% w/w organic solvent. The solvent is typicallyan organic volatile polar solvent, such as, without being limitedthereto, a solvent selected from the group consisting of acetone,isopropyl alcohol, acetonitrile, ethanol and methanol.

In some embodiments, trace amounts of alcohol are detected in the firstcomponent.

The particulate matter of the first component is unique in itscapability of forming a stable emulsion, once the particulate matter isbrought into contact with water. This is achieved, inter alia, due tothe presence of a surfactant in the first component. The surfactant isadded to the particles with the oil, before bringing the components intodryness.

In the context of the present disclosure, when referring to a stableemulsion it is to be understood as referring to dispersion of oil (thedispersed phase) in water (the dispersion medium) for a period of atleast 1 hour, at times, at least 2, 3, 4, 5, 10 or even 24 hoursfollowing the formation of the emulsion. In other words, the stabilityis determined by the lack of separation into an oil phase and a waterphase. The lack of separation may be determined by any means known inthe art, including visible inspection.

Without being bound by theory, it is the inventor's position that theincorporation of a surfactant in the particulate matter contributes tothe stability of the emulsion formed. This is also evident from thenon-limiting examples provided hereinbelow, where the use of potassiumsalts of fatty acids showed an advantage in terms of stability andsafety over other types of commercially available surfactants.

To form the emulsion, the particulate matter is mixed water. The amountof water depends on the amount of particulate matter. In someembodiments, for each gram of particulate matter (30% of which is oil),water is added to provide a one liter emulsion. As such, in a 1 literemulsion, 0.1 gr particulate matter provides an oil concentration of0.03% v/v). In some embodiments, the percentage of oil in the finalemulsion is in the range of 0.03% and 2% v/v.

In some embodiments, the mixing of the particulate matter with waterprovides an emulsion with a droplet size in the range of between 1 to 20μm and in some embodiments in the range between 3 to 10 μm.

In some embodiments, the emulsion is an anti-microbial emulsion.

The package contains a second component comprising at least oneantagonist of a microbial pathogen.

In some embodiments, the at least one antagonist of a microbial pathogenheld in a gel or gel-like carrier. Various materials may be used inorder to form the gel form for carrying the antagonists.

For example, the gel may be formed from a polysaccharide or combinationof polysaccharides with other substances.

According to some embodiments, the gel is selected from the groupconsisting of agar gel (agar-agar) Guar gum, gelatin, xanthan gum,methyl cellulose gel (cellulose gum), pectin base gel, gelatin gel, andothers, as known in the art.

In some embodiments, the second component comprises agar-agar therebyforming a gel holding the microbial antagonists.

In the context of the present disclosure, when referring to antagonistsof a microbial pathogen or a pathogen antagonist, it is to be understoodas a biological entity that inhibits the plant pathogen (a plantpathogen may also be referred to as a phytopathogen). Inhibiting, in thecontext of the present disclosure is to be understood as reducing growthof the pathogen by at least 50%, at least 70%, at least 90% or even byessentially eliminating the pathogen. The plant pathogen, in the contextof the present invention may be any prokaryotic or eukaryotic organism,including, without being limited thereto bacteria, a fungi, protozoa,nematodes, or any other disease causing parasite. As such, the microbialactivity of the at least one antagonist, may any one of antibacterial,antifungal, antiprotoxoal, antinematodal etc.

In some embodiments, the second component comprises at least oneantagonists. In some other embodiments, the second component comprises acocktail of antagonists. The cocktail is to be understood as acombination of two or more, at times, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15, antagonists combined together in the same or differentconcentrations.

In some embodiments, the at least one antagonist is of a type capable ofgrowing on sesame oil as a sole carbon source, as shown in Table 3hereinbelow. Such antagonists may be easily identified by conducting aconventional cultivation assay using sesame as the sole carbon sourceand identifying those cultivars that survived the experimental growingperiod.

In some embodiments, the antagonist may be referred to as abacteriostate, i.e., that slows down growth of organisms, and in someother embodiments, the antagonist may be referred to as a bactriocide,namely, that kills the organism.

In some embodiments, the at least one antagonist of a microbial pathogenis a soil born antagonist. In this context it is to be understood thatthe at least one antagonist may be obtained and isolated from the roots,soil and/or rhizophere of a plant that was shown to be tolerant (e.g.partially resistant) or resistant to the microbial pathogen.

In some other embodiments, the at least one antagonist of a microbialpathogen is a plant derived antagonist, e.g. isolated from a plant part,such as the leaves, the stem, the flower, the vascular system.

The at least one antagonist of a microbial pathogen may also be presentand thus derived from the soil (i.e. soil born) and from a plant part(e.g. the vascular system).

In accordance with some embodiments, the antagonists are of a pathogencausing infection in tomato plants.

In yet some embodiments, the antagonists are of the pathogen for whichtreatment is desired.

The second component may include one or more antagonists. In someembodiments, the second component includes a combination of severalantagonists in the same gel. However, in some other embodiments, whencombinations of antagonists are to be used, they are each carried by aseparate gel and mixed only prior to use. In other words, the secondcomponent is a combination of several “second components”, where eachantagonist is maintained separately and depending on the type ofpathogen to be treated, are combined prior to exposure of the plant.

When a combination of antagonists is used, the antagonists may beprovided/applied to the plant in the same amounts (CFU/ml) or indifferent amounts.

In accordance with some embodiments, the amount of an antagonist in thesecond component (either as a single antagonist or as a cocktail ofantagonists) may be in the range between 500 to 5,000CFU/ml/. The ratiobetween the antagonists, when used as a cocktail may vary, depending onthe type of pathogen to be treated and may be a priori determined byconventional laboratory methods, e.g. best bacteriostatic/bactriocidaleffect in a cultivation dish.

The type of antagonist will depend on the type of pathogen to be treatedby the package.

In some additional embodiments, the pathogen is Clavibactermichiganensis subsp. Michiganensis (CBM). In this embodiment, someantagonists that have been isolated from tomato plants that exhibitedtolerance to CBM are selected from the non-limiting group consisting ofPseudomonas species (Accession No. CBS133252), Pseudomonas alcaliphila(Accession No. CBS133254), Bacillus subtilis (Accession No. CBS133255),Pseudomonas cedrina (Accession No. CBS133256), Pseudomonas species(Accession No. CBS 133257), Pseudomonas species (AccessionNo.CBS133258), Pseudomonas spanius (Accession No. CBS133259).

Other antagonists are known in the art such as those provided in Table 4of the Report by the International Organization for Biological andIntegrated Control of Noxious Animals and Plants [Edited by Philippe C.Nicot 2011], the content of which is incorporated herein by reference.

In some other embodiments, antagonists of other plants may form part ofthe second component of the package disclosed herein. These may includeother agricultural crops, such as, without being limited thereto, thosederived from the Solanaceae family, e.g. tomato, pepper, eggplant,potato; from the Cucurbitaceae family, e.g. squash, melon, gourd,cucumber, pumpkin, luff and watermelons, but also any other seed bearingplants including horticultural plants, trees etc. The package typicallycomprises instructions for use of the first component and the secondcomponent to form an emulsion which is to be applied onto a plant. Theinstructions comprise, at least mixing the first component with thesecond component, optionally with the addition of an amount of water, toform an emulsion. In some embodiments, the concentration of the at leastone antagonist in the emulsion is at minimum 1,000 CFU/ml, or in therange of 500 CFU/ml to 5,000 CFU/ml.

In addition, antagonists may be found in various literatures, such as,without being limited thereto, the following, which are incorporatedherein by reference:

The infected The Pathogen plant The Antagonists Source of informationRalstonia Tomato, Bacillus megaterium, Journal of Plant PathologySolanacearum Pepper Enterobacter cloacae, 92(2): 395-406 (2010) Pichiaguillermondii and Candida ethanolica E. carotovora E-65 as a Bacillussp. The Scientific World subsp. and E-45 as a Journal (2012), Articlecarotovora Lactobacillus sp. ID 723293. P-138 Leptosphaeria canolaPseudomonas Biocontrol Science and maculans chlororaphis and P.Technology 16(5/6): 567582 aurantiaca (2006) Ralstonia Fungi inPseudomonas J. ISSAAS Vol. 18(1): 185- solanacearum peanut fluorescensRH4003 and 192 (2012) (Pseudomonas Bacillus subtilis AB89 solanacearum)Ralstonia Wilt Pseudomonas http://www.apsnet.org/publ solanacearumdisease of solanacearum isolates: ications/PlantDisease/BackI potatoB82; w163; wp95 and P. ssues/Documents/1983Arti fluorescenscles/PlantDisease67n05_49 9.pdf Rhizoctonia potato commercial productsof Crop Protection solani Bacillus subtilis (Kodiak) 24(11): 939-950,(2005) Actinomycetes Phytoprotection 82: 85-102 (2001) XanthomonasBacterial Streptomyces spp American Journal of oryzae pv. oryzae LeafBlight Agricultural and Biological Disease in Sciences 7(2): 217-223Rice (2012) Xanthomonas Isolates from soil and Rice Indstry, culture,and oryzae pv. oryzae water environment 549-553 Streptomyces PotatoPhytopathology 85: 261- spp scab 268 (1995); Can J Microbiol. 47(4):332-40 (2001) Sclerotinia Soybeans Bacillus MSU AgBioResearch 2011sclerotiorum, Potato amyloliquefaciens Annual Report, (2012)Streptomyces sp. scab (BAC03) URL: and Phytophthora Vegetablehttp://research.msu.edu/stor capsici crops ies/getting-root-soil-borne-diseases Rhizoctonia Black Egypt. J. Phytopathol., solani and Scurf Dry36(1-2): 45-56 (2008) Fusarium rot of sambucinum, Potato RhizoctoniaLettuce Endophytic strains, FEMS Microbiol Ecol solani Serratiaplymuthica 64: 106-116 (2008) 3Re4-18 and Pseudomonas trivialis 3Re2-7,rhizobacterium Pseudomonas fluorescens L13-6-12 Rhizoctonia PotatoPseudomonas Acta biol. Colomb. 12(1) solani fluorescens pages XXX (2007)Xanthomonas Tomato Rahnella aquatilis Microbiological Research,campestris pv. 160(4): 343-352 (2005) vesicatoria Erwinia PseudomonasPlant Disease 93(4): 386 amylovora (Fire fluorescens A506, URL: Blight)Pantoea agglomerans http://apsjournals.apsnet.or C9-1, and Pantoeag/doi/ pdf/10.1094/PDIS-93-4- 0386 Erwinia Erwinia herbicola ISHS ActaHorticulturae amylovora (Fire 117: II Symposium on Blight) FireblightURL: http://www.actahort.org/bo oks/117/117_21.htm Clavibacter Bacillussubtilis; BioControl 49: 305-313, michiganensis Rhodosporidium 2004subsp. diobovatum michiganensis

As noted above, in some embodiments, the antagonist is of a type that iscapable of growing on sesame oil as a sole carbon source.

At times, for preparing the emulsion, water may be added. When water isadded, the amount of water will depend on the amount of the firstcomponent. In some embodiments, for each gram of first component (e.g.30% of which are oil), water is added to provide one liter emulsion. Assuch, in 1 liter emulsion, 0.1 gr particulate matter provides an oilconcentration of 0.03% v/v.

In some embodiments, the percentage of oil in the final emulsion is inthe range of 0.03% and 2% v/v.

According to the above, a final formulation may be provided by a packagecontaining 20 grams of a powder of the first component (30% of which isthe oil), and 120 ml of an antagonist gel of the second component andthe instructions may include mixing the first component and the secondcomponent with water to form 20 liter emulsion containing 0.3%v/v ofoil.

Similarly, a package may contain 50 grams or 100 grams of a powder ofthe first component, and 120 ml of an antagonist gel of the secondcomponent and the instructions may include mixing the first componentand the second component with water to form a 50 or 100 liter emulsion.

In some embodiments, the second component is provided in the package inseparate units, each unit carrying a different antagonist in gel. Forexample, a package containing in total 120 ml gel, the gel mayconstitute several gel units, each gel unit carrying the same ordifferent antagonist, e.g. 8 gels of 15 ml gel each. This allowsselecting the types of antagonists to be used and changing thecombinations in the cocktail as needed, so as to provide a betterbiocontrol of the pathogen.

The package of the disclosure is suitable for the providence of a novelemulsion. In this respect, the present disclosure also provides anemulsion comprising particulate matter, a surfactant, at least onenatural oil and at least one bacterial antagonist, all as defined hereinas components of the package, wherein the at least one antagonist is ofa type that will grow on sesame oil as a sole carbon source.

The package disclosed herein is particularly suitable for use inpreventing or treating pathogenic infection in a plant. Treatment mayinclude inhibition of pathogen growth (bacteriostatic effect) as well astotal elimination of the pathogen infection (bacteriocidal effect).

In some embodiments, the treatment or prevention is of an infectioncaused by Clavibacter michiganensis subsp. Michiganensis (CBM).

In some embodiments, the treatment or prevention is of an infectioncaused by xanthomonas vesicatoria. In some other embodiments, thetreatment or prevention is of an infection caused by Streptomycesscabies.

In some embodiments, the treatment or prevention is of an infection in aplant belonging to the Solanaceae family, e.g. tomato, pepper, eggplant,potato. In one embodiment, the plant is a tomato plant.

In some embodiments, the package in accordance with the presentdisclosure, comprises

-   -   a carrier element comprising an essentially planar matrix        holding together one or more first compartments/wells for        carrying at least one first component comprising particulate        matter including at least one natural oil; and one or more        second wells/compartments for carrying at least one second        component comprising at least one antagonist of a microbial        pathogen; the one or more first wells and the one or more second        wells each being defined by a top opening and a recess extending        downwardly from the top opening;    -   a first film sealing the openings of the one or more first        wells; and    -   a second film sealing the openings of the one or more second        wells.

As appreciated, each well is a separate entity and mixing of content ofone well with the other is possible only after opening the sealingfilms.

A non-limiting example of a carrier element is illustrated in FIGS.1A-1D providing an isometric view (FIG. 1A) and top and side views(FIGS. 1B to 1D) of element 100 including a planar matrix 110 includinga single first well 120 and a plurality of, spaced apart, second wells130. In this non-limiting example, first well 120 is reversiblymountable in a depression 140 provided in planar matrix 110. In someother embodiments, a mountable well, such as well 120, may be fittedwithin an opening (hole) in the planar matrix 110 (not illustrated).Alternatively, the first well may be formed as an integral part ofplanar matrix 110 (not illustrated).

The plurality of second wells 130 are formed, in this particularembodiment, as an integral part of planar matrix 110. In an alternativeembodiment, any of second well(s) 130 may be of a kind that can bereversibly mountable in a depression 140, as illustrated for first well120 or in an opening provided in planar matrix (not shown). Inaccordance with this embodiment, first well 120 is in fact a type of acup that can be removed from the depression (in the form of a holdingwell) the planar matrix prior to use.

Carrier element 100 also includes a gripping unit 150, in the form of ahandle. The gripping unit may have other forms and shapes, so as tofacilitated stable and firm holding of the carrier element when thesealing films are pulled away and removed from the package so as torelease content of the first and second wells.

According to this non-limiting embodiment, inner diameter A of firstwell 110 that is configured for holding antagonist bacteria may be80-300 mm, and at times within the range between 100 mm-200 mm, or120-150 mm. Inner diameter B of second wells 130 which are configured tohold the same or different bacteria antagonist, is in the range of 20-40mm, at times within the range between 25-35 mm. The wells have typicallya depth of 20-60 mm, at times in the range of 20-30 mm.

In some embodiments, the total dimensions of the package are 500-550 mmlength, and 30-40 mm width.

FIGS. 1C, which is view of FIG. 1B also shows depression (recess orholding well) 140 for holding first well 120, depression 140 extendingdownwardly with respect to top side 160 of the carrier element. FIG. 1Dis another view of element 100 from side Y of FIG. 1B.

The first film and the second film may each separately cover and sealthe respective first wells and second wells. In accordance with someembodiments, and as also illustrated in FIGS. 2A and 2B, carrier element100 as illustrated in FIGS. 1A to 1D, is combined with a first film 170partially covering the opening of first well 120 and a second film 180superimposed over first film 170 and covering (sealing) second wells130. The first film 170 and the second film 180 may be fixedly attachedto each other such that upon pulling away second film 180, the firstfilm 170 is also pulled, thus allowing the opening of first wells 120and second wells 130 essentially simultaneously. However, in a similarmanner, first film 170 may be separate from second film 180 to allowindependent opening of the first wells 120 and second wells 130.

Turning now to FIGS. 3A-3E there is provided an element for use in apackage in accordance with another embodiment of the present disclosure.Therefore, for simplicity, like reference numerals to those used inFIGS. 1A-1D, shifted by 100 are used to identify components having asimilar function in FIGS. 3A-to 3E. For example, component 110 in FIG.1A is a well having the same function as well 210 in FIG. 2A.

Specifically, FIG. 2A is an isometric view, FIG. 3B is a top view andFIGS. 3C to 3E are side views of an element 200 from sides Z, X and Y,the element 200 having a general oblong (rectangular) shape, with theplanar matrix 210 including a single first well 220, being reversiblymountable in a depression 240 provided in planar matrix 210 and aplurality of, spaced apart, second wells 230. In accordance with thisnon-limiting embodiment, the first well 220 and the second wells 230 arepolygonal in shape, in this particular embodiment, quadrilateral.

Further, in accordance with this non-limiting embodiment, second well230 is shown as an integral part of the carrier element 200 but it mayequally be a releasable well, as shown in the non-limiting example ofFIG. 1A.

The dimensions of carrier element in accordance with this embodiment maybe different from that provided with respect to element 100, and in thisparticular embodiment may have total dimensions of 200-300 mm length,and 150-250 mm width, with the wells being deeper, i.e. having a depthin the range of between 50-60 nm.

As appreciated, the dimensions of the carrier element and in particularthe wells provided thereby may vary depending on the particularapplication of the package and the material to be carried thereby. Thoseskilled in the art would readily appreciate the adaptations required inthe dimensions in order to fit the carrier to the particularapplication.

As detailed above, the first well(s) of the package in accordance withthe present disclosure holds a composition of matter containing oil. Assuch at least the one or more first wells are formed from oil compatiblepolymers. When referring to oil compatible polymers it is to beunderstood as referring to polymers that are inert and do not change theproperties of the composite material including the natural oil. For thesame reason, the first film covering the first well(s) is composed ofoil compatible polymer.

In some embodiments, the first film of the respective first wells is orcomprises oil compatible thermoplastic polymers.

In some embodiments, it is required to maintain the composite materialin dry form. To this end, the first film is a fluid impermeable polymer.This are referred to in the art as high barrier (HB) films. HB films maybe defined by permeability to H₂O in the range of 3-4 g/m³/24 hr andpermeability to O₂ in the range of 6-8 cm³/m²/24 hr.

Some non-limiting examples of HB polymers that may form the first filmare those derived from any one of polyolefins, polyvinyls, polyesters.

The HB film is typically of a type that can easily peel-off the carrier.

In some embodiments, the first sealing film is a laminate having athickness in the range of 40-100 μm, at times, between 60-80 μm.

Turning to the one or more second wells, in accordance with someembodiments, since the second wells carry living matter, it is desirablethat the sealing film of these second wells, namely, the second film, begas permeable. In accordance with some embodiment, the second film ispermeable to oxygen or oxygen containing gas. To this end, thepermeability of the second film to H₂O may be in the range of 8-10g/m³/24 hr and permeability to O₂ of about 1,200 cm³/m²/24 hr.

The first and second films may be comprised of thermoplastic filmscomprising a single or blends of polymers selected from the groupconsisting of biaxially-oriented polyethylene terephthalate (BOPET),biaxially oriented polypropylene (BOPP), polyvinylidene chloride (PVDC),polyethylene polypropylene, polyvinyl alcohol.

In some embodiments, the films comprise a biaxial oriented polypropylene(BOPP). In some other embodiments, the films are a laminate of BOPP withanother polymer, such as polyethylene (PE). The laminate may comprisetwo, three of more laminated layers. The films to be used arecommercially available, e.g. from Glob-Plast (Plastart, Israel) andwidely used in the art.

The present disclosure also provides a method of providing a compositionfor treating or prevention of a pathogen infection in a plant, themethod comprise mixing a first component comprising particulate mattercomprising at least one natural oil, with a second component comprisingat least one antagonist of a microbial pathogen, and allowing saidmixture to form into an emulsion. The resulting emulsion may be referredto herein as an anti-microbial (e.g. bactericidal, fungicidal etc)effective emulsion.

In accordance with this method aspect, the first component and thesecond component are as defined herein.

Mixing may also require the addition of water to form the emulsion. Insome embodiments, the mixing provides an emulsion with a droplet size inthe range of between 1 to 20 μm and in some embodiments in the rangebetween 3 to 10 μm.

The present disclosure also provides a method of treating or preventinga pathogen infection in a plant, the method comprises applying to saidplant an amount of an emulsion comprising particulate matter, at leastone natural oil and at least one antagonist of a microbial pathogen. Inaccordance with this aspect of the present disclosure, the emulsion isobtained by mixing, preferably closely prior to application, a firstcomponent and a second component as defined herein.

In some embodiments, the method of treatment or prevention comprisesapplying the emulsion onto a plant. The application of the emulsion maybe by any means known in agriculture, including, without being limitedthereto, spraying the plant, irrigation.

In yet some other embodiments, the treatment or prevention may includeapplication onto the plant tubers, such as spraying of potato tubers (attimes referred to as low spraying of tubers).

The emulsion may be applied to the plant once to obtain theanti-microbial effect, or two or more times. When more than one dose isapplied, the doses may be provided with time intervals betweenapplications of between one day interval to more than one day (two,three and more days interval). The different doses may be the same ordifferent in the amount of the antagonists, the amount of the naturaloil or both, as well as the same or different in the type of antagonistsand/or natural oils provided.

In some embodiments, when more than one dose is applied, the variousapplications are in intervals of between 2 to 10 days at times between 3to 8 days.

In some embodiments, the plant is provided with at least 2, 3, 4, and attimes at least 5 doses of emulsion(s) provided in accordance with thepresent disclosure.

The frequency of treatment and the amount of doses depends on the typeof the infection (e.g. how violent/harmful/death threading it is), theseverity of infection, if already developed, the environmentalconditions, etc. Those versed in agriculture would be able to determinethe treatment schedule based on commonly used parameters.

For preventing an infection from development, it is required, inaccordance with some embodiments, to apply the emulsion to the plantfrom the day of planting of the plant or from a first day of a suspectedinfection.

For treatment of an infection, it is required, in accordance with someembodiments, to apply the emulsion to the plant from the day ofdetection of a suspected infection.

The present disclosure also provides for an isolated antagonisticbacterium selected from the group consisting of Pseudomonas species(Accession No. CBS133252), Pseudomonas alcaliphila (Accession No.CBS133254), Bacillus subtilis (Accession No. CBS133255), Pseudomonascedrina (Accession No. CBS133256), Pseudomonas species (Accession No.CBS133257), Pseudomonas species (Accession No.CBS133258), Pseudomonasspecies (Accession No. CBS134568), Pseudomonas spanius (Accession No.CBS133259), Pseudomonas mediterranea (Accession No. CBS134566),Pseudomonas chlororahis (Accession No. CBS134567) and Pseudomonasspecies (Accession No. CBS134568).

In some embodiments, the isolated antagonistic bacterium is within acarrier. In some embodiments, the carrier is a gel or gel-like carrieras defined hereinabove.

The isolated bacterium may have various applications. In someembodiments, the isolated bacterium, alone, or as a bacterial cocktailmay be used for treating or protecting plant against a disease caused bya pathogen, such as those disclosed hereinabove. The following are somenon-limiting examples for performing the invention. As will be shown,the formulations for the control of plant diseases the oils ingredientsand the ratio among the sesame oil or its substitutes and oregano oiland its substitutes are about constant (1:0.25(w/w) ratio of oreganooil:sesame oil) whereas the quantities of the other supplemented to theoils formulation are changed according to the plant, pest and theenvironmental conditions. At least one antagonistic bacterium out of thepossible bacteria is in use in combination with the oils formulation forthe control of plant diseases.

DESCRIPTION OF NON-LIMITING EXAMPLES

The study consisted of three parts: (i) isolation and multiplication ofthe pathogens and potential antagonists; (ii) in vitro screening ofpotential antagonists against Clavibacter michiganensis subsp.Michiganensis (CBM), and (iii) in vivo evaluation of selectedantagonists for the control of bacterial wilt disease on tomatoes.

Isolating of Pathogens and Potential Antagonistic Bacterial Strains

Tomato plants (Daniela, Hazera, Israel) were grown in greenhouses indouble rows of plants on each bed with a drip irrigation system. Theseedlings were planted in about 3 plants per meter row and werecultivated according the Dutch (Holland) method. The plots were undersurveillance for development of infection by CBM. Upon infection, plantsthat survived the infection were identified, and samples containing rootparts, soil (at depth of 30cm) and the rhizophere were taken intosterile bags and transported to the laboratory.

In the laboratory, samples of 10 grams each containing root parts, soil,and the rhizosphere were introduce into a stomacher bag containing 90 mlof agar solution 0.1% w/v (diluted with double distilled water (DDW),sealed and sterilized in an autoclave 121° C., 1 atm, 20minutes).Following sterilization, the samples were shaked for 2 hours at a speedof 20 rpm and room temperature. Then, the bags were transferred into astomacher device for 1 minute, at medium speed.

After stomaching the samples, each bag was opened by sterile scissors,and various dilutions of the samples were prepared in sterile tubescontaining each 9m1 of agar solution (0.1%w/v). The first dilution was 1ml sample into 9 ml agar (×10⁻² dilution), and continued up to ×10⁻⁸dilution.

From each diluted sample, 100 μl were transferred into Petri dishessupplemented with a media selected from one of the following agars:potato dextrose agar (PDA, Difco), a Nutrient Agar, Pseudomonas agar F(Difco) and incubated in an incubator (28° C.) for 5 days. Those ofinterest colonies were marked and transferred using a bacterial needleinto a fresh medium (the same as used for growing before isolation) toobtain an isolated single type of microbial colonies of potentialantagonists.

Multiplication of Isolated Antagonists

The microbial colonies suspected of having antagonistic effects werepurified from the microorganisms growing on the agar plates under alaminar flow hood and re-cultured in Erlenmeyer flaskon in a culturingbroth containing peptone (10 gr/litre), yeast extract (20 gr/litre),glycerol (10 gr/litre), MgSO₄ (0.1 gr/litre), CaCO₃ (2 gr/litre), Eachflaskon received a single isolated colony for cultivation as a pureantagonist and each flaskon was covered with a plastic cap, sterilizedin an autoclave for 20 min at 121° C., and kept at room temperature forone day.

Screening of Effective Antagonists of CBM and/or Xanthromonas.

Cultures showing antagonistic activity were screened for effective invitro suppression of the pathogen by transferring each of the suspectedantagonist using a bacterial needle into a Petri dish containing NAmedium (nutrient Agar medium), supplemented with 0.1% yeast extract and1% glycerol. The bacteria were placed on a straight line on the dish andthe dish was then incubated for 24 hours at 28° C. After incubationtime, CBM pathogen was seeded on an imaginary line perpendicular to thebacteria line and the dish was returned to incubation for an additionalperiod of 3 days. If the bacteria has antagonistic activity, a gapbetween the bacterial line and the pathogen line was formed during theincubation days those having the greater gap formed, were selected aspotential antagonists. These were further identified by the center ofCentraalbureau voor Schimmelcultures (Fungal Biodiversity Centre,Institute of Royal Netherlands Academy of Arts and Science (CBS-KNAW).

Table 1 provides the list of species deposited at the CBS-KNAW instituteon Nov. 19, 2012 and used in the final combination/cocktail. The specieswere stored at −80° C. in a glycerol solution (15%).

TABLE 1 Deposited antagonists Antagonist Accession No. Name Pseudomonasspecies CBS133252 BN12-27A Pseudomonas alcaliphila CBS133254 BN12-28Bacillus subtilis CBS133255 BN12-29 Pseudomonas cedrina CBS133256BN12-30 Pseudomonas species CBS133257 BN-12-31 Pseudomonas speciesCBS133258 BN12-32 Pseudomonas spanius CBS133259 BN12-33 Pseudomonasmediterranea AN1 CBS134566 BN13-01 Pseudomonas chlororahis AN10CBS134567 BN13-02 Pseudomonas species AN21 CBS134568 BN13-03

In addition, FIGS. 4A-4C show the effect of antagonists Pseudomonascedrina (CBS 1333256, “AN4”) (FIG. 4A) and Pseudomonas species (CBS1333258, “AN19”) (FIG. 4B) on the growth of CBM and Xanthromonas (twoseparate spreads/lines on the plate) as compared to the effect of acontrol being a non-antagonistic bacteria isolated from the same sourceof the antagonistic bacteria and grown on media without sesame oil(Control bacteria, FIG. 4C). Specifically, in FIG. 4A and FIG. 4B anon-growing zone is shown where the pathogen spreads of CBM andXanthromonas could not grow towards the antagonistic line. In thecontrol FIG. 4C, the pathogen spreads of CBM and Xanthromonas reachedthe control bacteria line. These results show that AN4 and AN19 haveantagonistic activity towards at least CBM and Xanthromonas.

The same experiment was conducted for each isolated bacteria which ledto the list of antagonistic bacteria of Table 1.

Preparing Antagonistic Microbial Gel

Isolated antagonists were separately transferred to Erlenmeyer flaskcontaining the medium used for multiplication (peptone (10 gr/litre),yeast extract (20 gr/litre), glycerol (10 gr/litre), MgSO₄ (0.1gr/litre), CaCO₃ (2 gr/litre) supplemented with 0.15% granulated Agar(Difco) and each antagonist at a concentration of between 10⁷ to 10⁸CFU/ml and shaked for 72 h at 28° C. The resulting gel like cocktail wasthen kept in gel form, at room temperature until use. It has been shownthat the antagonists can be preserved in this form for up to 12 monthswith a decrease in the bacterial population in logarithmic order of nomore than 2.

Determining Media for Maintaining the Antagonist

In order to determine the most appropriate media for storing theantagonistic bacteria, the following possible storage media were tested:

The Tested Antagonistic Bacteria:

The tested antagonistic bacteria were CBS 133252; CBS 133255 and CBS134567.

Each antagonistic bacteria was grown for 48 h on Petri dishes containingthe AN media. The colonies of each bacterium were collected from thesurface of the growing media into sterile distilled water to a finalconcentration of 10⁹CFU/ml.

The Tested Storage Media:

-   -   a. Distilled water (DW)    -   b. Oregano oil 79% and sesame oil 19% and DW 2%.    -   c. Oregano oil 4% and sesame oil 0.8% in DW.    -   d. An emulsion comprising oregano oil 4%, sesame oil 0.8% in        DW+0.05% Tween 80 (surfactant TWEEN® 80(Product Number: P4780        Brand: Sigma).    -   e. Antagonistic media (“AN media”) as a soft gel comprising        yeast extract (20 gr/litre), glycerol (10 gr/litre), MgSO₄ (0.1        gr/litre), CaCO₃ (2 gr/litre) supplemented with 0.15% granulated        Agar (Difco).

Preparing the Storage Media:

Each tested storage media (9 ml) was poured under aseptic condition into15 ml sterile tubes. In total 120 tubes for each tested storage media.

To each tube, 1 ml of the pre made antagonistic bacteria preparation(10⁹ cells/ml) was added, were inoculating to each tube to a finalconcentration of 10⁸ CFU of the tested bacteria in each tube. Theinoculated tubes were incubated for a period of 300 days at roomtemperature around 25° C.

Estimating the Survival of the Tested Bacteria in the Various StorageMedia:

During the 294 days of incubation, samples from each tube (5replications each) were taken periodically. From each tube a tenfolddilution in sterile distilled water was made from 1 to 10⁻⁸. Then, fromeach dilution, 100 micro liters were spared on the surface AN media.After incubation for 5 days at 25° C., the population was estimated asCFU/ml of the original test tube that were taken at a certain time. Theresults are the mean of the 5 replications at each time.

Results:

The survival results are summarized in Tables 2A-2E below:

TABLE 2A Survival of antagonistic bacteria in distilled water Days frominoculation CBS 133252 CBS133255 CBS133255 1 3.1 × 10⁸ 2.6 × 10⁸   1.8 ×10⁸ 14 1.6 × 10³ 1 × 10⁶  4 × 10³ 28 40  6 × 10³ 10  42 0 2 × 10² 0 56 020  0 70 0 0 0 84 0 0 0 98 — — — 112 — — — 126 — — — 140 — — — 154 — — —168 — — — 189 — — — 210 — — — 231 — — — 252 — — — 273 — — — 294 — — —

TABLE 2B Survival of antagonistic bacteria in Oregano oil 79%, sesameoil 19% and DW 2% Days from inoculation CBS 133252 CBS133255 CBS133255 16 × 103 8.5 × 103 1 × 103 14 0 20  0 28 0 0 0 42 0 0 0 56 — — — 70 — — —84 — — — 98 — — — 112 — — — 126 — — — 140 — — — 154 — — — 168 — — — 189— — — 210 — — — 231 — — — 252 — — — 273 — — — 294 — — —

TABLE 2C Survival of antagonistic bacteria in Oregano oil 4% and sesameoil 0.8% in DW Days after inoculation CBS 133252 CBS133255 CBS133255 12.4 × 10⁸ 2.5 × 10⁸ 1.7 × 10³ 14 0 10  0 28 0 0 0 42 0 0 0 56 — — — 70 —— — 84 — — — 98 — — — 112 — — — 126 — — — 140 — — — 154 — — — 168 — — —189 — — — 210 — — — 231 — — — 252 — — — 273 — — — 294 — — —

TABLE 2D Survival of antagonistic bacteria in an emulsion (oregano oil4%, sesame oil 0.8% in DW + 0.05% Tween 80) Days after inoculation CBS133252 CBS133255 CBS133255 1 2 × 10⁸ 3 × 10⁸ 2 × 10⁸ 14 0 30  0 28 0 10 0 42 0 0 0 56 0 0 0 70 0 0 0 84 — — — 98 — — — 112 — — — 126 — — — 140 —— — 154 — — — 168 — — — 189 — — — 210 — — — 231 — — — 252 — — — 273 — —— 294 — — —

TABLE 2E Survival of antagonistic bacteria in gel (yeast extract,glycerol, MgSO₄, CaCO₃, granulated Agar) Days after inoculation CBS133252 CBS133255 CBS133255 1 3.2 × 10⁸ 2.6 × 10⁸ 1.9 × 10⁸ 14 3.6 × 10⁸3.0 × 10⁸ 1.6 × 10⁸ 28 3.5 × 10⁸ 3.0 × 10⁸ 1.5 × 10⁸ 42 3.6 × 10⁸ 3.0 ×10⁸ 1.6 × 10⁸ 56 3.5 × 10⁸ 2.6 × 10⁸ 1.5 × 10⁸ 70 4.2 × 10⁷ 2.2 × 10⁸3.5 × 10⁷ 84 1.0 × 10⁷ 1.0 × 10⁸ 1.0 × 10⁷ 98 3.0 × 10⁶ 3.3 × 10⁷ 1.0 ×10⁶ 112 3.0 × 10⁶ 3.0 × 10⁷ 1.0 × 10⁶ 126 4.0 × 10⁶ 4.0 × 10⁷ 9.0 × 10⁵140 8.0 × 10⁵ 9.3 × 10⁶ 6.0 × 10⁵ 154 8.0 × 10⁵ 8.8 × 10⁶ 4.0 × 10⁵ 1686.0 × 10⁵ 6.0 × 10⁶ 5.0 × 10⁵ 189 3.0 × 10⁵ 3.0 × 10⁶ 1.0 × 10⁵ 210 1.0× 10⁵ 9.0 × 10⁵ 1.0 × 10⁵ 231 8.0 × 10⁴ 8.1 × 10⁵ 6.1 × 10⁴ 252 7.4 ×10⁴ 8.1 × 10⁵ 7.4 × 10⁴ 273 7.6 × 10⁴ 8.1 × 10⁵ 6.4 × 10⁴ 294 7.6 × 10⁴8.1 × 10⁵ 5.8 × 10⁴

Specifically, the results presented in Tables 2A to 2E show that thesurvival of the three tested antagonistic bacteria in the four firsttested storage media was significantly different from the gel basedmedia.

Specifically, only the gel (agar containing) media supported the longterm (294 days) survival of the antagonistic bacteria. At the end of theexperiment the population of CBS 133252 and CBS 133255 was above 5.0×10⁴CFU/ml, while CBS 133255 survived to a level of about 8.0×10⁵. In allother tested media the antagonistic bacteria did not survive after theabout 40 days.

Further Characterization of Antagonistic Bacteria

To further characterize the antagonistic bacteria, the difference ingrowth of the bacterial (as well as non-antagonists) on various carbonsource material has been investigated. Specifically, the growth of 10antagonistic bacteria and 6 non-antagonistic bacteria on sesame oil,glucose or glycerol as separate carbon sources was examined. A mediawithout carbon source was used as the control.

Materials

The growth media included:

Distilled water 500 ml NH₄H₂PO₄ 0.10 gr MgSO₄*7H₂O 0.01 gr KCl 0.10 grTested carbon source 4.20 gr

The Bacteria Stock Solution Included:

The bacteria was collected from 24 h bacterial culture from which asolution of 0.65 Absorbance at 480 nm, diluted 1:100 with distilledwater was prepared.

Method

Ten mililiters of the growth media with or without the tested carbonsource was poured into 30 ml Erlenmeyer flasks and inoculated with 100μl of the selected bacterial solution. The inoculated flasks wereincubated at 26° C., for 24 h and then subjected to a ten-fold dilutionand counted. Water was used as control.

Table 3 provides the colony forming units/ml for each texted carbonsource and bacteria. In Table 3, the isolated and deposited bacteriafrom Table 1 were compared with bacteria isolated from the soil asdescribed above, but found to have no antagonistic activity, these beingreferred to as Pseudomonas spp 12 (P. spp 12), Pseudomonas spp 13 (P.spp 13), Pseudomonas spp 14 (P. spp 14), Pseudomonas spp 15 (P. spp 15),Bacilus spp 36, E. coli 4.

The results in Table 3 show that bacteria with no identifiedantagonistic activity were not able to grow on sesame oil as a solecarbon source.

TABLE 3 Bacterial growth Bacteria deposit number Control* Sesame oilGlucose Glycerol CBS134566 60 5 × 10⁸  10⁷ 2 × 10⁹ CBS133252 80 3 × 10⁸ 10² 4 × 10⁸ CBS133254 100 1 × 10⁹ 6 × 10² 1 × 10⁹ CBS133255 50 6 × 10⁸7 × 10² 7 × 10⁸ CBS133256 110 9 × 10⁷ 6 × 10⁸ 5 × 10⁸ CBS134567 90 3 ×10⁸ 1 × 10⁹ 5 × 10⁸ CBS133257 130 2 × 10⁹ 120 4 × 10⁹ CBS133258 140 7 ×10⁷ 156 1 × 10⁸ CBS133259 60 1 × 10⁸ 150 1 × 10⁸ CBS134568 70 6 × 10⁷ 3× 10⁸ 7 × 10⁷ P. spp 12 60 75 4 × 10⁹ 8 × 10⁸ P. spp 13 70 54 3 × 10⁸ 1× 10⁸ P. spp 14 90 100 8 × 10⁸ 5 × 10⁸ P. spp 15 90 86 3 × 10⁹ 6 × 10⁸Bacilus spp 36 90 100 3 × 10⁹ 7 × 10⁸ E. coli 4 110 98 8 × 10⁸ 4 × 10⁸Water 0  0 0

Verifying Anti-Bacterial Effect of Essential Oils Materials and Methods

To verify the anti-bacterial effect of essential oil, the followingassay was conducted.

Oil: Oregano oil with the following particulars: country of origin:Bulgaria; plant parts: flowering plant; cultivation method: certifiedorganics, method of extraction: steam distilled.

Bacterial strains: Escherichia coli, Staphylococcus aureus, Salmonella,Clavibacter and Xanthomonas campestris were obtained from the collectionof Prof. G. Kritzman Israel.

Disc Diffusion method: Bacteria were grown in nutrient broth test tubesat 27° C. for 24 hrs. The paper discs were sterilized by autoclave inpreparation for the disc diffusion method. Each bacteria (100 μl), wasplaced on Nutrient agar (NA) plates and allowed to dry for 3-5 minutes.The paper discs were saturated in 100% concentration of the oreganoessential oil (20 ul), and then placed onto each NA plate freshly coatedwith bacteria. The positive control used was 3% H₂O₂ solution and thenegative control was DI water. The plates were incubated at 27° C. for48 hours. The zone of inhibition was measured by standard ruler.

Results

The anti-bacterial effect of the commercial organic oils on bacteria issummarized in Table 4, showing a greater inhibition zone for the oreganooils treated bacteria as compared to the controls.

TABLE 4 Anti-bacterial effect of oregano oil The tested bacteriaInhibition zone (mm) E. coli 15 S. aureus 19 Salmonela 21 Clavibacter 24Xanthomonas campestris 22 Positive control 8 Negative control 0

Preparation of Essential Oil Powder Materials

For preparing the oil powder, the following materials were used:

Natural oils:

Oregano oil 100% (essential oil) and Sesame oil 100% (carbon-base oil),both purchased from Makes Scents Natural SPA line, Lancaster Pa., USA.

Surfactants:

Thymol, Carvacrol, Tween80, Tween 65, Tween R85 and Egg Lecithin allpurchased from Sigma-Aldrich.

Span 40 purchased from Fluka, Israel.

Zohar LQ-215 (Potassium fatty acids) and Zohar PT-50 (Potassium fattyacids) purchased from Zohar Dalia.

Silica beads:

Tixosil (SiO₂) purchased from Rhodia group.

Aerosil 200 and Sipernat 50S (SiO₂, 20 μm) purchased from EvonikIndustries AG.

Solvent:

Acetone and Acetonitrile purchased from J. T. Becker, Isopropanol (IPA),Gadot.

Methods Powder Preparation

For laboratory scale production the powders containing the natural oils,surfactants and the silica beads were prepared using common labglassware set up including laboratory bottles of 20-50 ml sizes,spatulas, magnetic stirrers and heating plates. Generally, the naturaloil was weight and each was separately mixed with the selectedsurfactant in a 20 ml vial, to which the solvent was added. The mixtureof each oil were mixed and heated to a temperature of about 40° C. untilhomogeneous solutions were obtained. To the homogenous solutions thesilica beads were added until the liquid was absorbed by the beads. Thebottles were left in the fuming hood overnight until all solvent hasevaporated.

Loading of each of the oil in the final dry powders was 30-42%. The drypowders contained 2%-7% water.

All ratios of ingredients for powders preparation are provided in Tables5A-5D:

TABLE 5A Oregano oil based powder Surfactant Silica beads Oregano TweenTween Tween Span Aerosil Solvent Form. No. oil 80 Lecithin 85 65 40Tixosil 200 Acetone ORG-18A 0.5 g 0.5 g 0.1 g  0.8 g 1 g ORG-18B 0.5 g0.5 g 0.1 g  0.8 g 1 g ORG-18C 0.5 g 0.5 g 0.1 g  0.8 g 1 g ORG-18D 0.5gg 0.5 g 0.1 g  0.8 g 1 g ORG-20C 0.5 g 0.5 g 0.1 g 0.56 g 0.24 g 1 gORG-20D 0.5 g 0.5 g 0.1 g  0.4 g  0.4 g 1 g

TABLE 5B Sesame oil based powder Surfactant Silica beads Sesame TweenTween Tween Span Aerosil Solvent Form. No. oil 80 Lecithin 85 65 40Tixosil 200 Acetone SES-19A 0.5 g 0.5 g 0.1 g  0.8 g 1 g SES-19B 0.5 g0.5 g 0.1 g  0.8 g 1 g SES-19C 0.5 g 0.5 g 0.1 g  0.8 g 1 g SES-19D 0.5gg 0.5 g 0.1 g  0.8 g 1 g ORG-21C 0.5 g 0.5 g 0.1 g 0.56 g 0.24 g 1 gORG-21D 0.5 g 0.5 g 0.1 g  0.4 g  0.4 g 1 g

TABLE 5C Self emulsified Oregano oil based powder using anionicsurfactants Surfactant Solvent Zohar Zohar Silica beads Isopropyl Form.No. Oregano oil PT-50 LQ 215 Tixosil Aerosil 200 alcohol Acetone ORG-22A 0.5 g  0.5 g 0.56 g 0.24 g 0 ORG-22B  0.5 g  0.5 g  0.4 g  0.4 g   1 gORG-24A  0.5 g 0.25 g  0.4 g  0.4 g 0 ORG-24B  0.5 g 0.25 g  0.4 g  0.4g 0.5 g ORG-24C 0.75 g 0.25 g  0.4 g  0.4 g   1 g ORG-28  0.5 g 0.25 g0.56 0.24 g 1 g

TABLE 5D Self emulsified Sesame oil based powder using anionicsurfactants Surfactant Silica beads Solvent Zohar Zohar Isopropyl Form.No. Sesame oil PT-50 LQ 215 Tixosil Aerosil 200 alcohol Acetone ORG-23A 0.5 g  0.5 g 0.56 g 0.24 g 0 ORG-23B  0.5 g  0.5 g  0.4 g  0.4 g   1 gORG-25A  0.5 g 0.25 g  0.4 g  0.4 g 0 ORG-25B  0.5 g 0.25 g  0.4 g  0.4g 0.5 g ORG-25C 0.75 g 0.25 g  0.4 g  0.4 g   1 g ORG-29  0.5 g 0.25 g0.56 0.24 g 1 g

For greater amounts, laboratory electro-mechanical means similar tothose used in the industry were employed. These included:

-   -   1. Vertical Mechanical Stirrer DC Hsiangtai equipped with        propeller;    -   2. Peristaltic pump 4.4 Carter 4/6 cassette manostat and tubing;    -   3. Dynamic Exim 5 L powder mixer equipped with ribbon type        mixing blades    -   4. Balances    -   5. Beakers 1-2 L and containers 1-3 L

The preparation included weighting and mixing the oil with thesurfactant(s) in a 1L beaker, to which isopropyl alcohol was added whilemixing until a homogenous solution was obtained. The silica beads(Sipernat 50S) were added to a 2L beaker to which the homogenoussolution was slowly (rate of 10 ml/min) added while mixing (30 rpm)until all liquid was absorbed into the beads.

All ratios of ingredients for powders preparation are provided in Tables6A and 6B. The loading of the oil in the range of about 30%-42% wasmaintained.

TABLE 6A Oregano oil based powder Form. Surfactant Silica beads SolventNo. Oregano Oil Zohar PT 50 Sipernat 50S IPA 33 20 g 10 g 30 g 20 g 34200 g  100 g  300 g  200 g  37A 20 g 15 g 30 g 20 g 37B 20 g 10 g 300 g 15 g 38 2 × 2 × 2 × 2 × 200 g 125 g 300 g 125 g

TABLE 6B Oregano oil based powder Form. Surfactant Silica beads SolventNo. Oregano Oil Zohar PT 50 Sipernat 50S IPA 35 200 g 100 g 300 g 200 g39 200 g 125 g 300 g 125 g

In addition, also mixtures of powders (those containing Oregano oil andthose containing Sesame oil) were prepared. Specifically, 400 g offormulation ORG-34 (beads carrying oregano oil) was mixed with 100 g offormulation SES-35 (beads carrying Sesame oil) in Dynamic Exim 5 Lpowder mixer at 10 rpm producing the mix O&S-A.

Each type of oil based powder was dried in the vacuum oven at 40° C. for24 hr prior to mixing the two populations together.

In a different process, 800 g of formulation ORG-38 was mixed with 200 gof formulation SES-39 in Dynamic Exim 5 L powder mixer at 10 rpmproducing the mixed beads formulation O&S-B.

The mixed bead powders were used as is.

Characterization Determination of Water Content in Powder

Water content was determined using Mettler Toledo DL-38 Karl Fishertitrator according to USP <921> method.

Determination of Isopropanol Content in Powder

IPA content was determined using a headspace analysis according to theparameters bellow:

Gas chromatograph Agilent 7890 A Column BPX Volatiles, 60 m × 0.25 mm,1.4 μm, SGE Oven Program 45° C. for 2 min, then 10° C./min to 100° C.,then 25° C./min to 240° C., for 5 min. Split 1:25 Mass spectrometerAgilent 5975C Autosampler program CTC Combi PAL Pre-incubation time: 300s Incubation temp.: 80° C. Syringe temp: 100° C. Volume of injection:500 μl Headspace vial 20 ml Volume of sample (water) 2 ml Calibrationpoints (μg/ml) 10, 25, 100, 500, 1000 Concentration of ISTDs 50 μg/ml(ethanol)Assay of Oregano Oil in Dry Powder using HPLC

Impurities profile were determined in accordance with the methodreported by H. Hajimehdipoor “A validated high performance liquidchromatography method for the analysis of thymol and carvacrol in Thymusvulgaris L. volatile oil” in Pharmacogn Mag. 2010 July-September; 6(23):154 158 and adopted by SoluBest. For this purpose Nucleosil 100 C18 HD,3μ, 150×3 mm column and Ultimate 3000 Dionex (Germany) HPLC system withphotodiode array (PDA) detectors and Chromeleon Version 6.80 softwarepackages were used. The mobile phase is Acetonitrile:Water (50:50, v/v).Minimum resolution between Carvacrol and Thymol peaks is 1.5.

Standard solutions were prepared in duplicate as following:

About 3 mg Thymol and 20 mg Carvacrol were weighted into 50 mLvolumetric flask, and dissolved in 40 mL of diluents, then brought up tovolume with the diluent and mixed. The resulting concentration of theThymol standard solution was about 0.06 mg/mL and Carvacrol standardsolution was about 0.4 mg/mL.

Sample solutions were prepared in duplicate as following:

About 70 mg of powdered sample was weighted into a 25 mL volumetricflask, then brought up to volume with the acetone and mixed.

Assay of Sesame Oil in Formulation using GC

Sesame oil absorbed on silica beads was trans-methylated overnight withmethanolic HCl solution at 60° C. Heptadecanoic acid, used as aninternal standard, was added to beads before derivatization. Methylesters of fatty acids were extracted with hexane and dried overanhydrous sodium sulfate prior to GC analysis.

Calibration standards were prepared from different concentrations ofsesame oil and blank beads. Conditions of derivatization and amount ofinternal standard were the same as described in sample preparation.

Quantitative analysis of sesame oil in beads was performed using Agilent7890 gas chromatograph equipped with FID detector. Compounds wereseparated on DB-23 capillary column.

Results

Conventional HPLC and GC analytical methods for Oregano and Sesame oilsassay were employed.

The chromatograms of the tested powders showed that no degradation(according to the conventional markers, Thymol and Carvacrol aromaticcompounds) of the oil was caused during the powder preparation andstorage. Table 7 below provides % of Oregano oil and Sesame oil,respectively, in the powder based on Thymol and Carvacrol aromaticcompounds analysis by HPLC.

TABLE 7 Oregano oil content in formulations as measured by HPLC Form. NoSample % via Thymol % via Carvacrol ORG-18A ORG-18A-1 25.2 27.1ORG-18A-2 27.1 28.3 Average 26.1 27.7 Difference, % 7.2 4.3 ORG-28ORG-28-1 33.8 34.9 ORG-28-2 32.1 33.2 Average 32.9 34.1 Difference 5.55.2 ORG-32 ORG-32-1 28.0 30.3 ORG-32-2 28.1 30.7 Average 28.0 30.5Difference 0.6 0.7 ORG-34 ORG-34-1 26.9 29.8 ORG-34-2 27.7 30.1 Average27.3 30.0 Difference 2.1 2.0 ORG-38 ORG-38-1 28.8 28.7 ORG-38-2 28.029.3 Average 28.4 29.0 Difference 4.1 2.0

Table 8 provides the % of Sesame oil in the formulation as determined byGC

Chromatograph.

TABLE 8 Sesame oil content in formulations as measured by GCchromatography Form. No. Sample % via C16:0 % via C18:0 % via C18:2SES-19A SES-19A-1 — — 28.0 SES-19A-2 — — 27.6 Average — — 27.8Difference, % — — 1.4 SES-35 SES-35-1 29.8 30.5 37.5 SES-35-2 30.6 30.633.4 Average 30.2 30.6 35.5 Difference, % 2.6 0.3 12.3 SES-39 SES-39-132.8 33.0 39.8 SES-30-2 31.5 32.3 37.4 Average 32.2 32.7 38.6Difference, % 4.1 2.2 6.4

The water content measured using Karl Fisher titration found that thepowder contains 5-7% of water. It appears the source of the water isfrom Zohar PT 50 surfactant, which contents 50% of water.

As to IPA content, GC Headspace precise analysis demonstrate the IPAcontent in the formulations, which is summarized in Table 9.

TABLE 9 IPA content in the powders Form. No Sample Amount (gr.) IPA(μg/ml) IPA (%) 34 19.6 1,265 12.9 35 200 1,139 11.4 38 20.3 1,117 11.039 19.8 492 5.0

As can be seen from the Table 9, the amount of IPA varies from 5 to 13%.However, in the field, the formulations were diluted for at least 30times and as such, the content of IPA was reduced to 0.17-0.43%, whichis negligible and very safe amount.

The different types of dry powders prepared showed stable after longterm (more than a year) storage. In addition to the above, it is notedthat the powders have a characteristic odor. The oregano oil basedformulations have off-white color and sesame oil based powders arewhite.

Upon contact with water tested formulations (ORG-28 and SES-29immediately form an emulsion, which were stable for 24 h. The emulsionsconsisted of droplets of 3-10 microns. The spray-ability of theemulsions was good without clogging the filters.

Safety studies in the field showed that the tested oil based powderswere safe. This was determined by the presence (or not) of burns on theplants, as determined by conventional phytotoxicty parameters.

Further, long term (8 weeks) stability of the powders was determined.Specifically, the Oregano and Sesame based powders were separatelysealed in aluminum foil bags and placed at accelerating storingconditions (40° C. for 8 weeks). Assay of Oregano oil was measured viatwo major constituents—Carvacrol and Thymol-in the beginning of thestability study (initial point) and after 8 weeks using HPLC-UVtechnique. The obtained values were normalized to the amounts of markersin the pure oregano oil.

Assay of sesame oil was measured via two major constituents - C16:0 andC18:0 in the beginning of the stability study (initial point) and after8 weeks using GC-FID analysis of methylated fatty acids. Transmethylation was performed upon acidic catalysis (with MeOH/HCl) usingC17:0 as an internal standard. The obtained values were normalized tothe amounts of markers in the pure sesame oil.

No significant changes were observed in the both formulations: amount oforegano and sesame oils were similar before and after stability studies.

Water content was tested using Karl Fisher method. The amount of waterwas reduced on 42% after 8 weeks of storing in accelerating conditionsin both formulations.

Isopropanol content was tested using GC method. The amount of IPA wasreduced on 36% after 8 weeks of storing in accelerating conditions inoregano formulation, but it was preserved in the sesame formulation.

Without being bound by theory, it appears that the containers were leakyand in order to reduce water or IPA loss, the containers may be morehermetically sealed.

Powders stored 8 weeks at 40° C. showed good ability to form a stableemulsion similar to those of the initial powders. The stabilitymeasurements are summarized in Table 10 below:

TABLE 10 Stability Assays Sesame oil (%) Time Oregano oil (%) via viaWater, IPA, point via C16:0 via C18:0 Thymol Carvacrol % % Oreganopowder Initial 28.4 29.0 7.05 11 SORG-121-38 8 weeks 29.8 29.1 4.05 7Sesame powder Initial 32.2 32.7 6.65 5 SES-121-39 8 weeks 33.3 34.2 3.815Solubilization and Anti-Bacterial Activity with Different Surfactants

In order to create stable oil-in-water emulsion a surfactant(emulsifying agent) with HLB of 8-20 is required. Thus, in thefollowing, two surfactants were tested Tween 80 having an HLB value of15 and potassium salt of fatty acids extracted from palm, coconut,olive, castor and cottonseed plants (potassium salt oleate having an HLBvalue of 20).

It has been found that with the potassium salts of fatty acids the ratioof oil/surfactant required for obtaining a stable emulsion of oreganooils is 1:0.4 while with Tween 80, the required oil/surfactant ratio was1:1.

The correlation between HLB values and solubilization capacity of eachsurfactant was found in the current case of Oregano oil, i.e. bettersolubiilztion with potassium salts of fatty acids. Isopropanol was usedas process aid compound, which also provided additional stability forproducing emulsions.

For anti-bacterial effect, several emulsifiers were tested with oreganooil and sesame oil, in water.

The tested emulsifiers included: Tween 20; Tween 80; Triton X 100;Lecithin; SDS; Sodium Stearate and Potassium fatty acid. Eachemulsifiers was tested at the following concentrations (in percentage)1;5;10;15;20 for a mixture of water containing 25% oregano oil with 5%sesame oil.

The stability during the first 24 hours (i.e. lack of phase separation)and anti-bacterial activity of each emulsion were determined. Antibacterial activity was determined by measuring the inhibition zones of20 μl emulsion towards the following plant pathogenic bacteria: Clavibacter; Xanthomonas and Streptomyces spp. and by spraying the emulsionson pepper plants as test plants for phytotoxicity symptoms.

The results showed that potassium fatty acid was the most suitablematerial in creating stable emulsion at 10% concentration; theinhibition zone of the potassium salt of fatty acid was greater than theother tested emulsions and had no phytotoxicity on pepper plants. Thedata obtained (not shown) clearly demonstrated phytotoxicity on pepperplants when the emulsifiers were Tween 20 Tween 80, Triton X100 or SDS,as the plants sprayed with the emulsions created therewith died, whilebeing very vital when sprayed with the sodium stearate potassium fattyacid.

In a different set of experiments, the oregano oil and surfactant wereabsorbed on silica beads (with the air of isopropanol, as describedabove). Good anti-bacterial results were obtained when 0.5% oforegano-based powder was dispersed in water. This powder contained about25% of oregano oil and 10% of potassium fatty acids emulsifier, namely,a concentration of 0.125% oregano oil in emulsion was active enough andneeded only 0.05% of surfactant to be solubilized from the powder form.

Surprisingly, even a lower concentration of powder was sufficient toproduce good crop protection in a greenhouse study. Only 0.2% of powderand consequently 0.05% of oregano oil was enough to disperse in water(to form an emulsion) and showed excellent and repeatable anti-bacterialeffect with the plants being treated therewith remaining completelyvital as compared to infected, but untreated control.

Preparation of Biocontrol Cocktails

Three biocontrol cocktails were prepared as follows:

The antagonistic gels containing a cocktail of the bacteria listed inTable 1 above, were each used at an amount 10⁷-10⁸ CPU/ml from eachantagonist.

Each of the antagonistic gels were then used in two concentrations, thefirst as obtained, i.e. with no dilution (at an amount 10⁷-10⁸ CPU/mlfrom each antagonist) mixed with beads carrying 0.03% natural oil(equivalent to 1 gr beads in 1 liter water) referred to by theabbreviation NB, and a second formulation diluted ×2 with water to formformulation NBx2 in which the concentration of the oil is 0.06%.

Biocontrol Experiments

In the bio-control assays, the following oil powder composition wasemployed:

Materials and compositions

Oils: Sesame oil 100% (SPAline Lot sicP1A11/01); Oregano oil 100%(SPAline Lot 0015181)

Surfactant: Zohar PT-50 (Zohar Dalia, Batch 05511PM1142);

SiO₂ particles: Sipernet 50S (20 μm, Evonik Industries, lot 1462)

Alcohol: Isopropanol (IPA, Gadot).

Formulation:

Oregano Sesame Zohar Sipernat Amount oil oil PT-50 50S IPA Total Kg 4010 31 75 21 177 % 22.6 5.6 17.5 42.4 11.9 100

Preparation of Powder

First, the oregano oil and sesame oil were mixed, to which IPA was addeduntil a homogenous solution was obtained. To the solution, thesurfactant was added by mixing until a non-viscous homogenous solutionwas obtained. The solution was sprayed on the SiO₂ powder and mixing wasperformed using low sheer force equipment until all liquid was absorbedand continued for an additional period of 15-30min. Then the powder wasgrind to breakdown aggregates and sieved through mesh 1,000 μm. Forstorage, the bag containing the powder was hermetically sealed andstored. Storage was for at least 2 years, at 15-30° C.

Biocontrol Example I In vitro Effects of Biocontrol Cocktail on VariousPathogens

Tested pathogens: the following four antagonistic bacteria were tested(identified by the deposit accession number): CBS 133252, CBS 133254,CBS 134567 and CBS 133259.

Clavibacter, Xanthomonas campestris pv. Vesicatoria and Yeast—were takenfrom the private collection of the inventor.

Method:

Pathogen preparation—Each of the tested pathogens were separately platedon an yeast extract dextrose agar (YDC) and grown for 48 h at 25±1° C.,after which the surface of each plate was washed with sterile distillwater. The pathogen was then collected into tubes containing steriledistilled water. For the testing, the two tested pathogens were combinedand plated on a petri dish.

Antagonist preparation—The antagonist was prepared by spreading a sample(100 μl) of one of the above listed test antagonists, on the surface ofa Petri dish containing YDC media and growing the antagonist for 48 h at25±1° C. on Antagonistic media (the agar based gel disclosed herein).Then, with a cork cutter, pieces of the media including the antagonistcocktail were removed and placed on the surface of plates carrying thetwo pathogens for combined incubation.

After 24 h of incubation at 25±1° C. of the pathogen together with eachantagonist, an inhibition zone around the pieces of the antagonists werecreating. The inhibition zones were exhibited by a halo around colonies.FIG. 5A shows the growth of pathogen bacteria without treatment with anantagonist, FIG. 5B shows the effect of CBS 133252, FIG. 5C shows theeffect of CBS 133254, FIGS. 5D shows the effect of CBS 133567 and FIG.5E shows the effect of CBS 133259

Biocontrol Example 2 Effects of Biocontrol Cocktail on Tomato Wilt

In vivo evaluations were conducted in three net greenhouses to assessthe antibacterial activity of the three biocontrol cocktails on tomatoDaniella plants. The tomato plants were grown with trellises.

As the positive control, the Copper Hydroxide (Kocide, Milchan Bros) wasused at a concentration of 0.3% (the Copper control)

Non-treated plants were used as Negative Control.

All plants were watered with either 50m1 of a biocontrol cocktail, theCopper Control or with none (Negative Control) for three consecutivedays after planting, followed by spraying the plants every 7 days. Forspraying the essential oil powder (in the respective amount) and theantagonistic gel were mixed directly in the container of a backpackmotor sprayer and the entire plant, including leaves and stems, weresprayed.

After 2 weeks, the second plant in each growing row in the greenhousewas infected with CBM inoculum comprising two CBM strains isolated frominfected plants grown in the Besor area of Israel. The inoculum included2 strains of CBM, strain number 32 (strain designation 189/1-1) andstrain number 42 (stain designation 189/6-1) [Frida Kleitman et al.Characterization of Clavibacter Michiganensis Subsp. Michiganensispopulation in Israel Eur. J. Plant Pathol (2008) 121:463-475]

Inoculation was performed by creating a cut in a leaf of the plant, nearthe stem, and dipping the leaf in the CBM inoculum.

During growing, the plants were under surveillance and plants that wereinfected were observed.

Statistical Test

The results were analyzed using the Student's T-test.

Results

The above field trial was repeated six times, and the results of the sixrepetitions are summarized in Table 11A, providing mortality rate of thetomato plant, and Table 11B providing infection level by CBM, as testedusing Agria kit for CBM (Cmm ImmunoStrip Test catalog no. STX 44001Agdia Incorporated 30380 County Road 6 Eelkhart, Ind. 46514 USA)

TABLE 11A Survival rate of tomato plants Treatment Mortality RateNegative Control 18%  Copper Control 8% NB 2% NBx2 1%

TABLE 11B Percent of CBM infection in tomato plants Treatment CBMinfection Negative Control 83.5 ± 11 Copper Control 58.8 ± 15 NB 50.5 ±14 NBx2 25.8 ± 12

Table 12 provides statistical comparison between the results of Table11A.

TABLE 12 Statistical comparison in mortality rate between treatmentgroups Compared Groups P < 0.05 No treatment vs. NBx2 Yes No treatmentvs. NB Yes No treatment vs. Copper formulation No Copper formulation vs.NB Yes NB vs. NBx2 Yes

In the Table “YES” indicates that there is a statistical significantdifference; while “NO” indicating that there is no statisticalsignificant difference Table 9 shows that when comparing NB to any othertreatment group, the positive effect is statistically significant. Whencomparing Copper Control to Negative Control, the positive effect of theCopper Hydroxide treatment is insignificant. Doubling the dose(concentration of NB vs. NBx2) show also a statistically significantincreased effect.

In addition, Table 13 and FIG. 6 show the effect of treatment on plantdeath.

TABLE 13 mortality Treatment % Mortality Negative Control 18%  CopperControl 8% NB 2% NBx2 1%

Specifically, Table 13 shows that non-treatment, Negative Control Group(control) and the Copper Group (Cu) have higher percentage of mortalityas compared to the treatment groups NB and NBx2. In each group, the siderow was more sensitive than the centered row (not shown). The differencebetween the side rows and centered rows may be attributed to thelocation of the side rows in the greenhouse, being affected by externalparameters.

The results also show synergism. Table 14 shows that at a widetemperature range the combination of the natural oils (sesame oil beadsand oregano oil beads) with the antagonists increase the survival of theplants to a much greater extent than the effect of each component alone[(A+B)/C>1].

Specifically, the results show that the combined effect of antagonistsonly (A) and oils only (B), divided by the Control (C) is greater than1, i.e. synergism.

TABLE 14 Synergism % of Antag- % of % ONLY the onist the % of Un- Un-Antag- Con- and Con- Only Con- treated treated Temp. onist trol Oilstrol Oils trol (Con- (Con- A + ° C. (″A″) (″A″) (″C″) (″C″) (″B″) (″B″)trol) trol) B/C 18 3.33* 60 0 0 3.3 66 5 100 Yes 20 14.2 65.4 0 0 15.872.8 21.7 100 Yes 25 35.8 44.8 4.2 5.3 64.2 80.25 80 100 Yes 28 44.2 455 5.1 66.7 67.9 98.3 100 Yes 32 39.2 53.5 0 0 54.2 73.9 73.3 100 Yes

Biocontrol Example 2 Effects of Biocontrol Cocktail on Pepper

For the prevention of pepper infection by xanthomonas vesicatoria (XV),the same biocontrol cocktail used in Example 1 is used, with the sametreatment methodology. The pathogen was originally isolated by theinventor from diseased pepper plants and pepper seedlings. The isolatesof xanthomonas vesicatoria (XV), are in the collection of G. Kritzman.

Biocontrol Example 3 Effects of Biocontrol Cocktail on Potato

For the prevention of potato infection by Streptomyces spp., the causingagent of Streptomyces scabies., a biocontrol cocktail comprising threeantagonists isolates obtained according to the procedure provided abovewith some of the previous list, were used. The three antagonists areprovided in Table 15 below.

TABLE 15 Deposited Antagonists for potato treatment Antagonist AccessionNo. Name Pseudomonas mediterranea AN1 CBS134566 BN13-01 Pseudomonaschlororahis AN10 CBS134567 BN13-02 Pseudomonas species AN21 CBS134568BN13-03

The biocontrol cocktails are prepared as described above, at threeconcentrations 1% (no dilution); 0.5% (1:1 dilution) and 0.025% (1:4dilution) active ingredient (of the oil formulation) with a constantconcentration of the bacterial antagonists at a final concentration inthe tank mix of 10³ CFU/ml.

Before use, the antagonists are mixed in a mixing tank with one of thedry formulations contained among others ingredient Organo oil dry powderand Sesame oil dry powder (80:20 ratio as described above). The potatoseeds tubers are entered into a spraying cell by rolling on a conveyer.In the cell the tubers pass through a cloud of drops (about 80u indiameter each). After each tuber is rolled about 8 times around its axisthe treated tubers are collected in a container as dry treated tubers.The treated tubers now are coated with a fine layer of the completeformulation are ready to be planted not before 72 h after the treatment.

In the case of the potato scab, the efficiency of the treatment istested not by artificially inoculating tubers but by choosing potatoseeds lots naturally high infested with many typical symptoms on eachseed tuber. It could be of the common scab type or of the deep pittedscab symptoms. The seeds are examined in two procedures:

a) Samples of 60 tubers in replications are taken to the laboratory. Inthe laboratory all the tubers are peeled by a commercial potato peelerwhich is operated without watering the tubers during the peelingprocedure. A large sample of the potato peels are collected. Ten gramsare taken to a Stomacher bag containing 90 ml of 0.1% (w/v) water agarsupplemented with 0.05% (w/v) ascorbic acid. This suspension is mixedfor 2 h on a rotary shaker and then for 1 min in stomacher. After thishomogenization a tenfold dilutions is done and aliquots of 100 μl wereinoculated on the surface of Petri dishes containing semi selectivemedia for isolation of Streptomyces. After 5 incubation days at 28° C.the CFU/gr peel is calculated for the treated potato seeds and incomparison with the untreated control.

b) The treated and the untreated potato seeds are planted in a trialfield with at least a 4 replication each. In such experiment the growthand yield parameters as well as the scab control on the daughter tubersat the harvest time are evaluated.

The population of Streptomyces per gram of peel was calculated and theresults are in Table 16:

TABLE 16 treatment of Potato scab Oil component Streptomyces spp.dilution CFU/gr peel No dilution (1% w/v) 40 1:1 3 × 10² 1:2 1 × 10⁴Control (untreated) 1 × 10⁷Further biocontrol Experiments

For testing treatment or prevention of other pathogenic infections, thefollowing protocols may be used.

I. Protocol for In Vitro Antagonistic Activity

Transfer each antagonistic bacteria in a separate Petri Dish (9 cm)using a bacteriological transfer loop to form a single antagonist linealong the diameter of the dish, and incubate the dish for 48 h at 28° C.

Transfer a tested pathogen along a line perpendicular to theantagonistic line without touching the walls of the dish.

Incubate the dishes for a further period of 5 days at a temperaturebetween 25° C. and 28° C.

Measure the zone at which the antagonist inhibited growth of thepathogen (the “inhibitory zone”). The size of the inhibitory zone beingindicative of the level of antagonistic activity of the bacteria.

II. Affect of Oil Formulation without Antagonist.

Create a stock solution of the oil formulation (described above, withoutthe antagonistic bacteria) by dissolving the oil powder in the mediumthat is suitable for culturing the tested pathogen bacteria (culturemedium which includes agar that is liquid at 37° C.) at a finalconcentration of oil of 1% w/v. From the stock solution, create a seriesdouble diluted concentrations with the culture medium (dilutions of 1:1,1:2, 1:4, 1:16, 1:32, 1:64, 1:128, 1:256 and 1:512) of the oilformulation. Introduce into Petri dishes and allow the oil containingmedium to harden.

Prepare pathogen suspension by dilution with saline colonies after 48 hof cultivation and calibrate pathogen concentration in the suspension toreach an absorbance of 0.6AU at 480 nm.

Dilute the pathogen bacteria up to a concentration of 10⁻⁸/ml and plate100 μl on the hardened oil containing culture media.

After 5 days of incubation at 25° C-28° C. of each plate at thedifferent oil concentrations, determine the number of colonies.

III. Bactericide or Bactriostat Activity

Prepare antagonist formulations with different amounts of oil diluted asdescribed above, albeit with sterilized water and each diluted oilformulation with the antagonists at constant concentration of 10³antagonists per ml.

An amount of 9 ml antagonists formulations with the different oildilutions was incubated in a tube with an aliquot of 1 ml of a testedpathogen suspension in a concentration of 10⁸CFU/ml. The pathogensuspension was prepared from cultures after 72 h cultivation.

After incubation for 3 h, the content of each tube is filtered on a 0.45mm mesh membrane with sterilized water after which the pathogen iswashed off from the membrane using sterilized water and vortexing.

The washed off bacteria are then tenfold diluted in a series up to aconcentration of 10⁻⁸ CFU/ml. From each dilution 100 μl aliquots areplatted on a Petri dish with a suitable culture media and cultured. Theformation of colonies is indicative of bacteriostat activity of theformulations while absence of colonies is indicative of bacteriocidalactivity.

IV. Plant Testing:

The plant testing is conducted in a pot or in a green-house. Plants withat least 5 leaves are used for testing.

In a pot:

On day 1, the plant is sprayed with the tested oil/antagonistformulation at various concentrations and after 12 h the plant issprayed with the pathogen solution being at a concentration of about 10⁶CFU/ml.

Two hours after infection, the plants are once again sprayed with thesame oil/antagonist formulation again and is continuously sprayed every6 days for a period of 6 weeks. After 6 weeks, the disease incidence isestimated and recorded.

In an Open Test Field or Greenhouse:

The plants are grown using the conventional method of their planting andgrowing in the field of greenhouse.

After planting, the plants are irrigated with an amount of about 100m1oil/antagonist formulation /plant and then each plant is sprayed withthe same formulation, with continuous spraying every 5 -6 days.

The disease incidence is estimated and recorded.

Toxicology Tests

Various toxicological tests were performed using the formulationincluding a cocktail of all deposited antagonists (Table 1) each at aconcentration of 10⁹ as follows:

(i) Acute Oral Toxicity in the Rat:

Protocol:

Female Sprague-Dawley™ (SD™) rats were divided into two groups (n=3),each receiving a single dose of 2000 mg/kg of the tested formulation(Dosage Volume of 1.97 ml/kg, oral gavage (PO) administration), at aninterval of administration between the two groups of 24 hours.

Results:

-   -   No mortality occurred in any of the tested rats throughout the        14 days observation period.    -   No noticeable clinical signs in reaction to dosing were evident        in any of the animals during the immediate post-dosing time or        throughout the entire 14-day observation period.    -   Body weight gain at the end of the 14-day study period of all        animals was found to be within range of normally expected        values.    -   No gross pathological findings were evident in any of the        animals at the time of their scheduled necropsy.    -   Based on the lack of adverse reactions following a single oral        administration to the rats at the limit test dose level of 2000        mg/kg, the tested formulation may be regarded as not        representing an acute toxic risk by this route of administration        and the acute oral median Lethal Dose (LD50) is greater than        2000 mg/kg.

(ii) Acute Dermal Toxicity

Protocol:

A single dose level of 2000 mg/kg of the tested formulation wastopically applied for 24-hr exposure duration to a 5 female and 5 maleSprague-Dawley (SD™) rats. The rats were observed for a total of 14days.

Results:

-   -   No mortality occurred in any of the rats throughout the entire        14-day study period.    -   No noticeable clinical signs in reaction to dosing were evident        in any of the rats throughout the entire 14-day observation        period.    -   Body weight gain of all rats at the end of the 14-day study        period was found to be within the range of normally expected        values.    -   No gross pathological findings were evident in any of the rats        at the time of their scheduled necropsy    -   Based on the lack of adverse reactions following a single dermal        application to the rats the tested formulation was as not        representing an acute toxic risk by this route of administration        and the estimated acute dermal median lethal dose (LD50) was        determined to be greater than 2000 mg/kg.

Acute Dermal Irritation/Corrosion in Rabbits

Protocol:

The potential skin irritation effects of the tested formulation wereassessed following a single dermal application to a group of 3 male NZWrabbits, according to the testing procedure recommended by the OECDGuideline for the Testing of Chemicals, Section 4, No. 404, “AcuteDermal Irritation/Corrosion” adopted 24 Apr. 2002.

Specifically, all rabbits were topically applied with a single sample ofabsorbent gauze measured 2×3 cm each moistened with 0.5 ml of the testedformulation for a 4 hour exposure duration. Each gauze patch was held incontact with the skin with non-irritant irritant tape and a suitablesemi-occlusive dressing (TUBIGRIP stockinet) to retain the gauze patchesand the tested formulation throughout the exposure period.

Dermal reactions were scored and recorded at the standard time points of1, 24, 48 and 72 hours after patch removal.

Results:

-   -   No erythema or edema was noted throughout the 72-hour study        period in any of the rabbits.    -   No other dermal reactions were noted in any of the rabbits        throughout the study period.    -   No noticeable clinical signs in reaction to treatment were        evident in any of the rabbits throughout the entire study        period.    -   No abnormal changes in body weight were noted in any of the        rabbits throughout the entire study period.    -   Consideration of the calculated Primary Irritation Index (PII)        was 0, led to the conclusion that irritation response is        categorized as negligible.        (iii) Skin Sensitization (Local Lymph Node Assay)

Protocol:

The potential of the tested formulation to cause skin sensitization wasassessed on the basis of the testing procedures by the OECD Guidelinefor the Testing of Chemicals, Section 4, No. 429 “Skin Sensitization:Local Lymph Node Assay”, adopted 22 Jul. 2010.

Specifically, three dilutions of the tested formulation were prepared inPhysiological Saline. Pluronic® L92 (1% v/v) was added to each dosingsolutions prior to application.

In order to ensure reproducibility and sensitivity of the testprocedure, a well-known weak to moderate contact allergen, 25% HCA, wasincluded in this study, diluted with the Negative Control used.

Three groups of BALB/c female mice (n=5) were subjected to applicationof the tested formulation (one dilution per group) once daily for threeconsecutive days, at a dose volume of 25 μl applied on the dorsum ofeach outer ear. Additional two equally sized groups were subjected toapplication of either the Negative Control (Physiological Saline) or thePositive Control (25% HCA), under identical conditions.

Five days after the first topical application, all mice were injectedwith 3H-Methyl Thymidine by intravenous (IV) injection. Approximately 5hours later, all mice were euthanized and the auricular lymph nodes wereexcised. A single cell suspension of both left and right lymph nodescells from individual animals was prepared.

The incorporation of 3H-Methyl Thymidine was measured by a β-Counter andexpressed as Disintegration Per Minute (DPM)/animal.

The Stimulation Index (SI) was calculated for the groups treated withthe tested formulation dilutions and the Positive Control. The SI valuesfor all dilutions were lower than 3 and the SI values for the PositiveControl was higher than 3.

Results:

-   -   No mortality occurred in any of the mice in all groups        throughout the 5-day study period.    -   No noticeable clinical signs in reaction to treatment were        observed in any of the tested formulation or Negative Control        treated mice throughout the 5-day study period. All Positive        Control treated mice displayed redness at the ears.    -   All mice showed an increase in body weights at the end of the        5-day study period.    -   Under the conditions of the study and according to the        calculated Stimulation Index values, it was concluded that the        tested formulation did not cause reactions associated with skin        sensitization.

(iv) Acute Eye Irritation/Corrosion in Rabbits

Protocol:

The potential eye irritation/corrosion effects of the tested formulationwere assessed following a single eye instillation to a group of 3 maleNZW rabbits, according to the testing procedure recommended by the OECDGuideline for the Testing of Chemicals, Section 4, No. 405, “Acute EyeIrritation/Corrosion” adopted Oct. 2, 2012.

Initially, single dose of 0.1 ml the tested formulation was applied tothe right eye of one rabbit (Initial Test) and subsequently to twoadditional rabbits (Confirmatory Test). The left eye of each rabbit wasnot treated and served as control.

Ocular reactions were scored and recorded at the standard time points of1, 24, 48 and 72 hours following application.

Results:

-   -   No ocular reaction was noted throughout the 72-hour study period        in any of the rabbits.    -   No noticeable clinical signs in reaction to treatment were        evident in any of the rabbits throughout the entire study        period.    -   No abnormal changes in body weight were noted in any of the        rabbits throughout the entire study period.    -   Under the conditions of this study, it was concluded that the        tested formulation did not cause reactions associated with eye        irritation/ corrosion.

1. A package comprising: i) at least one first component comprisingparticulate matter comprising at least one natural oil and at least onesurfactant; and ii) at least one second component comprising [[an]] atleast one antagonist of a microbial pathogen,. wherein the at least onefirst component and the at least one second component are contained inseparate compartments of said package.
 2. The package of claim 1,wherein said first component comprises the particulate matter in anessentially dry form.
 3. (canceled)
 4. (canceled)
 5. The package ofclaim 1, wherein: (a) said particulate matter comprises silica (SiO₂);or (b) said particulate matter has a size distribution in the range of10-25 μm; or (c) said particulate matter exhibits at least one of asurface area in the range of 400-500 m² N₂/g and oil capacity in therange of 300-350 DBP/100 gram particulate; or (d) said natural oil isabsorbed in said particulate matter; or any combinations of (a)-(d). 6.(canceled)
 7. (canceled)
 8. The package of claim 1, wherein said naturaloil comprises at least one essential oil.
 9. (canceled)
 10. (canceled)11. The package of claim 1, comprising either (i) particulate mattercarrying at least one essential oil and at least one carbon-richnutrient oil, or (ii) two or more populations of particulate matter,wherein each population of particulate matter carries a different typeof natural oil, and at least one population of particulate mattercarries an essential oil, and at least one population of particularmatter carries carbon-rich nutrient oil.
 12. (canceled)
 13. (canceled)14. The package of claim 1, wherein the particulate matter comprisebetween 20% to 50% w/w natural oil out of the total weight of theparticulate matter. 15-21. (canceled)
 22. The package of claim 1,wherein said second component is in a form of a gel.
 23. (canceled) 24.(canceled)
 25. The package of claim 1, wherein said at least oneantagonist of the microbial pathogen is a soil born bacteria or abacteria isolated from a plant part, wherein the plant exhibitstolerance or resistance to the microbial pathogen.
 26. (canceled) 27.The package of claim 1, wherein the at least one second componentcomprises an antagonist at a concentration of between 500 CFU/ml to5,000 CFU/ml.
 28. (canceled)
 29. The package of claim 1, furthercomprising two or more second components, each of said second componentscarry a different type of antagonist or a cocktail of antagonists. 30.The package of claim 1, wherein the at least one antagonist is abacteria selected from the group consisting of Pseudomonas species(Accession No. CBS133252), Pseudomonas alcaliphila (Accession No.CBS133254), Bacillus subtilis (Accession No. CBS133255), Pseudomonascedrina (Accession No. CBS133256), Pseudomonas species (Accession No.CBS133257), Pseudomonas species (Accession No. CBS133258), Pseudomonasspecies (Accession No. CBS134568), Pseudomonas spanius (Accession No.CBS133259), Pseudomonas mediterranea (Accession No. CBS134566),Pseudomonas chlororahis (Accession No. CBS134567), and Pseudomonasspecies (Accession No. CBS134568). 31-35. (canceled)
 36. The package ofclaim 1, further comprising: one or more first wells holding said atleast one first component; one or more second wells holding said atleast one second component; wherein the one or more first wells and theone or more second wells each have a top opening and a recess extendingdownwardly from said top opening, and the wells are held together in anessentially planar matrix; and a first film sealing the openings of theone or more first wells and a second film sealing the openings of theone or more second wells.
 37. The package of claim 36, wherein at leastone of said one or more first wells and one or more second wells areeither (i) integrally formed in said planar matrix or (ii) reversiblymountable in an opening or recess within said planar matrix. 38-40.(canceled)
 41. The package of claim 36, wherein said first film is anoil compatible and fluid impermeable thermoplastic polymeric film. 42.(canceled)
 43. The package of claim 36, comprising a single first welland a plurality of second wells, each second well comprises the same ordifferent antagonist or a cocktail of antagonists.
 44. The package ofclaim 36, comprising a single second film sealing said of one or moresecond wells, wherein the second film is permeable to gas; said secondfilm is superimposed over said first film and optionally fixedlyattached to said first film. 45-52. (canceled)
 53. A method of treatingor preventing a pathogen infection in a plant, wherein the methodcomprises applying to said plant an amount of an emulsion,. wherein theemulsion comprises particulate matter, at least one natural oil, atleast one surfactant and at least one antagonist of a microbial pathogenthat causes said pathogen infection. 54-60. (canceled)
 61. The method ofclaim 1, wherein the at least one second component comprises a mixtureof antagonists of a Clavibacter michiganensis subsp. Michiganensis(CMB), wherein the CBM is selected from the group consisting ofPseudomonas species (Accession No. CBS 133252), Pseudomonas alcaliphila(Accession No. CBS 133254), Bacillus subtilis (Accession No. CBS133255), Pseudomonas cedrina (Accession No. CBS 133256), Pseudomonasspecies (Accession No. CBS 133257), Pseudomonas species (Accession No.CBS 133258), Pseudomonas species (Accession No. CBS 134568), Pseudomonasspanius (Accession No. CBS 133259), Pseudomonas mediterranea (AccessionNo. CBS 134566), Pseudomonas chlororahis (Accession No. CBS134567) andPseudomonas species (Accession No. CBS134568).
 62. An isolatedantagonistic bacteria, wherein the isolated antagonistic bacteria isselected from the group consisting of Pseudomonas species (Accession No.CBS133252), Pseudomonas alcaliphila (Accession No. CBS133254), Bacillussubtilis (Accession No. CBS133255), Pseudomonas cedrina (Accession No.CBS133256), Pseudomonas species (Accession No. CBS133257), Pseudomonasspecies (Accession No. CBS133258), Pseudomonas species (Accession No.CBS134568), Pseudomonas spanius (Accession No. CBS133259), Pseudomonasmediterranea (Accession No. CBS134566), Pseudomonas chlororahis(Accession No. CBS134567) and Pseudomonas species (Accession No.CBS134568), optionally within a carrier.
 63. (canceled)